Bruton&#39;s tyrosine kinase as anti-cancer drug target

ABSTRACT

Receptor protein kinases (RPTKs) transmit extracellular signals across the plasma membrane to cytosolic proteins, stimulating formation of complexes that regulate key cellular functions. Over half of the known tyrosine kinases are implicated in human cancers and are therefore highly promising drug targets. A large-scale loss-of-function analysis of tyrosine kinases using RNA interference in the clinically relevant Erb-B2 positive, BT474 breast cancer cell line showed that Bruton&#39;s tyrosine kinase (BTK), a cytosolic, non-receptor tyrosine kinase that has been extensively studied for its role in B cell development, is required, in altered form, for BT474 breast cancer survival. This alternative form contains an amino-terminal extension that is also present in tumorigenic breast cells at significantly higher levels than in normal breast cells.

This invention was made with government support by U.S. Army MedicalResearch Acquisition Activity grant DAMD17-02-1-0729 and NCI R01CA136658to DSC. The government has certain rights in the invention.

FIELD

Embodiments of the invention find application in the field of cancertherapy.

BACKGROUND

Protein tyrosine kinases (PTKs) mediate the reversible process oftyrosine phosphorylation, providing the signals that activate or blocksignal transduction pathways that govern cell survival decisions and assuch are tightly regulated. Genes that regulate extracellular growth,differentiation and developmental signals are commonly mutated incancers. Perhaps it is not surprising therefore that PTKs comprise thelargest group of dominant oncogenes. Thirty of the 58 receptor proteintyrosine kinases (RPTKs) have been implicated in human cancer(Blume-Jensen and Hunter, 2001). Less than half of the cytoplasmicprotein tyrosine kinases have been associated with tumorigenesis, duenot to a less critical role in signal transduction regulation, however,but from an experimental bias that has focused on viral counterparts togain insight into potential transforming mechanisms (Blume-Jensen andHunter, 2001).

In recent years there has been a surge in efforts to discover genescritical to cancer signaling pathways that when inhibited would providespecific anti-cancer therapies (Lu and Chu, 2008) (Sabbah et al., 2008).Trastuzumab, (Herceptin), a humanized monoclonal antibody thatspecifically inhibits the HER2/neu/ErbB-2 (hereafter referred to asErbB-2) receptor tyrosine kinase, which is amplified and/orover-expressed in 25-30% of metastatic breast cancers, was the firsttargeted therapy to be approved by the FDA. As a single-agentmonotherapy, however, the primary response rate to trastuzumab is low,(12% to 34%) and the rate of primary resistance high, between 66% to 88%(Nahta and Esteva, 2006). Notably, however, the time to diseaseprogression, response rate and overall survival increase whentrastuzumab is used in combination with paclitaxel or docetaxel (Nahtaand Esteva, 2006). Indeed, recent successes in targeting moleculesintegral to survival pathways in combination with traditionalchemotherapeutics has led to significant efforts to identify new drugtargets that sensitize the breast cancer cell towards cell death(MacKeigan et al., 2005); (Call et al., 2008). Such additional drugtargets, specific to or over-expressed in breast cancer cells comparedto normal tissues, and known to be functionally relevant, are stillneeded, as are cancer-specific markers for use in detecting ordiagnosing cancer.

SUMMARY

In one embodiment, the instant invention provides a method of treatingcancer, comprising: a) providing a subject with cancer (e.g. breastcancer cells) and an inhibitor of a gene encoding a cytoplasmic tyrosinekinase, and b) treating said subject with said inhibitor. In a preferredembodiment, the cytoplasmic tyrosine kinase is a member of the Tecfamily of cytoplasmic tyrosine kinases and, in a more preferredembodiment, the cytoplasmic tyrosine kinase is Bruton's Tyrosine Kinase.In another embodiment, the cytoplasmic tyrosine kinase is a variant ofBruton's Tyrosine Kinase comprising an amino-terminal extension. Theamino acid sequence of the variant is SEQ ID NO. 1. The amino-terminalextension is SEQ ID NO 2. In one embodiment the extension comprises anadditional 34 amino acids. In one embodiment, the method of treatingcancer comprises treating with an inhibitor that comprises aninterfering RNA. Preferably, the treatment with the RNA results inreduced proliferation of the breast cancer cells. In one embodiment, theamino acid sequence of the variant and amino-terminal extension is SEQID NO. 3.

In one embodiment, the instant invention provides a method of diagnosingcancer, comprising: a) providing cells suspected to be breast cancercells and a ligand capable of binding to a variant of Bruton's TyrosineKinase, said variant comprising an amino-terminal extension; b)contacting said cells with said ligand under conditions wherein saidvariant is detected. In one embodiment, the amino-terminal extension ofthe variant used to diagnose cancer comprises an additional 34 aminoacids. In a preferred embodiment the ligand used binds to a portion ofthe 34 amino acid extension. In one embodiment, the ligand comprises anantibody or a fragment thereof.

In another embodiment, the invention provides a composition comprising avariant of Bruton's Tyrosine Kinase comprising an amino-terminalextension, the extension preferably comprising an additional 34 aminoacids.

In another embodiment, the invention provides a ligand-protein complexcomprising an antibody bound to the variant of Bruton's Tyrosine Kinase.

In yet another embodiment, the invention provides a kit for diagnosingcancer, the kit comprising a ligand capable of binding to a variant ofBruton's Tyrosine Kinase and instructions for its use.

In one embodiment, the present invention contemplates a compositioncomprising a purified variant of Bruton's Tyrosine Kinase comprising anamino-terminal extension. In one embodiment, the extension comprises anadditional 34 amino acids. In one embodiment, the present inventioncontemplates a ligand-protein complex comprising antibody bound to thepurified variant of Bruton's Tyrosine Kinase comprising anamino-terminal extension. In another embodiment, the variant comprisesthe amino acid sequence set forth in SEQ ID NO: 3. In yet anotherembodiment, the variant comprises an amino acid sequence at least 95%identical to SEQ ID NO: 3 that prevents apoptosis in a cancer cell.

In one embodiment, the present invention contemplates an isolated cDNAcomprising SEQ ID NO: 2.

In one embodiment, the present invention contemplates an interferingdouble stranded RNA that is at least partially complementary to SEQ IDNO: 2 that inhibits expression of a protein encoded by SEQ ID NO: 2. Inone embodiment, the present invention contemplates an interfering doublestranded RNA (siRNA) having a sense strand comprising the nucleotidesequence 5 ′-GGU UAU UGG AUG CCC AUU AUU-3 ′ (SEQ ID NO: 66). In oneembodiment, the present invention contemplates an interfering doublestranded RNA having an antisense strand comprising the nucleotidesequence 5 ′-UAA UGG GCA UCC AAU AAC CUU -3 ′ (SEQ ID NO: 67). In oneembodiment, the present invention contemplates an interfering doublestranded RNA having a sense strand comprising the nucleotide sequence 5′-CAA CAA AUG GUU AUU GGA UUU -3 ′ (SEQ ID NO: 68). In one embodiment,the present invention contemplates an interfering double stranded RNAhaving an antisense strand comprising the nucleotide sequence 5 ′-AUCCAA UAA CCAUUU GUU GUU -3 ′ (SEQ ID NO: 69).

In one embodiment, the present invention contemplates an isolatedantibody that specifically binds to the polypeptide of the amino acidsequence set forth in SEQ ID NO: 3. In another embodiment, the presentinvention contemplates an isolated antibody that specifically binds tothe polypeptide of an amino acid sequence at least 95% identical to SEQID NO: 3. In yet another embodiment, the present invention contemplatesan isolated antibody that specifically binds to a fragment of the aminoacid sequence set forth in SEQ ID NO: 3. In a further embodiment, thefragment consists of the C-terminal amino acids of the amino acidsequence set forth in SEQ ID NO: 3. In still further embodiments, thefragment consists of the 34 C-terminal amino acids of the amino acidsequence set forth in SEQ ID NO: 3. In additional embodiments, theantibody is a monoclonal antibody. In yet another embodiment, theantibody is a humanized antibody. In yet another embodiment, theantibody is an antibody fragment. In yet another embodiment, theantibody is labeled.

In one embodiment, the present invention contemplates a method oftreating cancer, comprising: a) providing: i) subject with cancer (e.g.breast cancer), ii) a chemotherapeutic agent, and iii) an inhibitor of agene encoding a cytoplasmic tyrosine kinase; and b) treating saidsubject with said chemotherapeutic agent and said inhibitor. In oneembodiment, the cytoplasmic tyrosine kinase is Bruton's Tyrosine Kinase.In one embodiment, the cytoplasmic tyrosine kinase is a variant ofBruton's Tyrosine Kinase comprising an amino-terminal extension. Inanother embodiment, the extension comprises an additional 34 aminoacids. In one embodiment, the inhibitor comprises an interfering doublestranded RNA. In one embodiment, the chemotherapeutic agent comprisesDoxorubicin or analogues thereof. In another embodiment, treating withsaid chemotherapeutic agent and said inhibitor results in reducedproliferation of the breast cancer cells within said subject. In anotherembodiment, said interfering double stranded RNA comprises a sensestrand having the nucleotide sequence of SEQ ID NO: 66. In anotherembodiment, said interfering double stranded RNA comprises an antisensestrand having the nucleotide sequence of SEQ ID NO: 67. In yet anotherembodiment, said interfering double stranded RNA comprises a sensestrand having the nucleotide sequence of SEQ ID NO: 68. In yet anotherembodiment, the interfering double stranded RNA comprises an antisensestrand having the nucleotide sequence of SEQ ID NO: 69. In furtherembodiments, said inhibitor comprises a mixture of interfering doublestranded RNAs comprising a sense strand having the nucleotide sequenceof SEQ ID NOs: 66 and 68 and an antisense strand having the nucleotidesequence of SEQ ID NOs: 67 and 69. In additional embodiments, thechemotherapeutic agent includes, but is not limited to, AC (Adriamycin,cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin,vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin,cyclophosphamide, vincristine, procarbazine, prednisone), BEP(bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF(cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV(cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide,Adriamycin, vincristine, prednisone), Ch1VPP/EVA (chlorambucil,vincristine, procarbazine, prednisone, etoposide, vinblastine,Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine, Adriamycin,dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin orplatinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil,Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin,adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone),MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin,cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin),ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide, etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin, cyclophosphamide,etoposide, cytarabine, bleomycin, vincristine, methotrexate,leucovorin), VAD (vincristine, Adriamycin, dexamethasone), Regimen I(vincristine, Adriamycin, etoposide, cyclophosphamide) and VAPEC-B(vincristine, Adriamycin, prednisone, etoposide, cyclophosphamide,bleomycin).

In one embodiment, the present invention contemplates a method oftreating cancer, comprising: providing: i) a subject with cancer (e.g.breast cancer), ii) a chemotherapeutic agent, and iii) an inhibitor of agene encoding a cytoplasmic tyrosine kinase, b) treating said subjectwith said chemotherapeutic agent, c) identifying resistance of at leastsome of said breast cancer cells to said chemotherapeutic agent; and d)treating said subject with said inhibitor. In one embodiment, thecytoplasmic tyrosine kinase is Bruton's Tyrosine Kinase. In oneembodiment, the cytoplasmic tyrosine kinase is a variant of Bruton'sTyrosine Kinase comprising an amino-terminal extension. In anotherembodiment, the extension comprises an additional 34 amino acids. Inanother embodiment, the inhibitor comprises interfering double strandedRNA. In yet another embodiment, the chemotherapeutic agent comprisesDoxorubicin or analogues thereof. In yet another embodiment, treatingwith the chemotherapeutic agent results in reduced proliferation of atleast some breast cancer cells within the subject. In one embodiment,the interfering double stranded RNA comprises a sense strand having thenucleotide sequence of SEQ ID NO: 66. In one embodiment, the interferingdouble stranded RNA comprises an antisense strand having the nucleotidesequence of SEQ ID NO: 67. In one embodiment, the interfering doublestranded RNA comprises a sense strand having the nucleotide sequence ofSEQ ID NO: 68. In one embodiment, the interfering double stranded RNAcomprises an antisense strand having the nucleotide sequence of SEQ IDNO: 69. In one embodiment, the inhibitor comprises a mixture ofinterfering double stranded RNAs comprising a sense strand having thenucleotide sequence of SEQ ID NOs: 66 and 68 and an antisense strandhaving the nucleotide sequence of SEQ ID NOs: 67 and 69. In anotherembodiment, the inhibitor results in reduced proliferation of at leastsome breast cancer cells within said subject identified as resistant tosaid chemotherapeutic agent. In yet another embodiment, thechemotherapeutic agent includes, but is not limited to, AC (Adriamycin,cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin,vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin,cyclophosphamide, vincristine, procarbazine, prednisone), BEP(bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF(cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV(cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide,Adriamycin, vincristine, prednisone), Ch1VPP/EVA (chlorambucil,vincristine, procarbazine, prednisone, etoposide, vinblastine,Adriamycin), CVAD/H erCVAD (cyclophosphamide, vincristine, Adriamycin,dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin orplatinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil,Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin,adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone),MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin,cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin),ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide, etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin, cyclophosphamide,etoposide, cytarabine, bleomycin, vincristine, methotrexate,leucovorin), VAD (vincristine, Adriamycin, dexamethasone), Regimen I(vincristine, Adriamycin, etoposide, cyclophosphamide) and VAPEC-B(vincristine, Adriamycin, prednisone, etoposide, cyclophosphamide,bleomycin).

In one embodiment, the present invention contemplates a method oftreating cancer, comprising: a) providing: i) a subject with cancer(e.g. breast cancer), ii) a chemotherapeutic agent, and iii) aninhibitor of a gene encoding a cytoplasmic tyrosine kinase, b) treatingsaid subject with said inhibitor; and c) after step b), treating saidsubject with said chemotherapeutic. In one embodiment, the cytoplasmictyrosine kinase is Bruton's Tyrosine Kinase. In another embodiment, thecytoplasmic tyrosine kinase is a variant of Bruton's Tyrosine Kinasecomprising an amino-terminal extension. In yet another embodiment, theextension comprises an additional 34 amino acids. In still furtherembodiments, the inhibitor comprises interfering double stranded RNA. Inone embodiment, the chemotherapeutic agent comprises Doxorubicin oranalogues thereof. In another embodiment, treating with thechemotherapeutic agent results in reduced proliferation of the breastcancer cells within the subject. In one embodiment, the interferingdouble stranded RNA comprises a sense strand having the nucleotidesequence of SEQ ID NO: 66. In one embodiment, the interfering doublestranded RNA comprises an antisense strand having the nucleotidesequence of SEQ ID NO: 67. In one embodiment, the interfering doublestranded RNA comprises a sense strand having the nucleotide sequence ofSEQ ID NO: 68. In one embodiment, the interfering double stranded RNAcomprises an antisense strand having the nucleotide sequence of SEQ IDNO: 69. In another embodiment, the inhibitor comprises a mixture ofinterfering double stranded RNAs comprising a sense strand having thenucleotide sequence of SEQ ID NOs: 66 and 68 and an antisense strandhaving the nucleotide sequence of SEQ ID NOs: 67 and 69. In yet anotherembodiment, the inhibitor results in reduced proliferation of the breastcancer cells within said subject. In yet another embodiment, thechemotherapeutic agent includes, but is not limited to, AC (Adriamycin,cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin,vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin,cyclophosphamide, vincristine, procarbazine, prednisone), BEP(bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF(cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV(cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide,Adriamycin, vincristine, prednisone), Ch‘VPP/EVA (chlorambucil,vincristine, procarbazine, prednisone, etoposide, vinblastine,Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine, Adriamycin,dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin orplatinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil,Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin,adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone),MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin,cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin),ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide, etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin, cyclophosphamide,etoposide, cytarabine, bleomycin, vincristine, methotrexate,leucovorin), VAD (vincristine, Adriamycin, dexamethasone), Regimen I(vincristine, Adriamycin, etoposide, cyclophosphamide) and VAPEC-B(vincristine, Adriamycin, prednisone, etoposide, cyclophosphamide,bleomycin).

In one embodiment, the present invention contemplates a method oftreating cancer, comprising: a) providing: i) a subject with breastcancer cells, at least some of said breast cancer cells exhibitingresistance to a chemotherapeutic agent, and ii) an inhibitor of a geneencoding a cytoplasmic tyrosine kinase, and b) treating said subjectwith said inhibitor. In one embodiment, the cytoplasmic tyrosine kinaseis Bruton's Tyrosine Kinase. In one embodiment, the cytoplasmic tyrosinekinase is a variant of Bruton's Tyrosine Kinase comprising anamino-terminal extension. In one embodiment, the extension comprises anadditional 34 amino acids. In one embodiment the inhibitor comprisesinterfering double stranded RNA. In another embodiment, treating withthe inhibitor results in reduced proliferation of at least some of thebreast cancer cells within the subject. In another embodiment, theinterfering double stranded RNA comprises a sense strand having thenucleotide sequence of SEQ ID NO: 66. In yet another embodiment, theinterfering double stranded RNA comprises an antisense strand having thenucleotide SEQ ID NO: 67. In yet another embodiment, the interferingdouble stranded RNA comprises a sense strand having the nucleotidesequence of SEQ ID NO: 68. In another embodiment, the interfering doublestranded RNA comprises an antisense strand having the nucleotidesequence of SEQ ID NO: 69. In one embodiment, the inhibitor comprises amixture of interfering double stranded RNAs comprising a sense strandhaving the nucleotide sequence of SEQ ID NOs: 66 and 68 and an antisensestrand having the nucleotide sequence of SEQ ID NOs: 67 and 69. In oneembodiment, the inhibitor results in reduced proliferation of at leastsome breast cancer cells within the subject identified as resistant tothe chemotherapeutic agent. In another embodiment, the chemotherapeuticagent is selected from the group consisting of AC (Adriamycin,cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin,vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin,cyclophosphamide, vincristine, procarbazine, prednisone), BEP(bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF(cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV(cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide,Adriamycin, vincristine, prednisone), Ch1VPP/EVA (chlorambucil,vincristine, procarbazine, prednisone, etoposide, vinblastine,Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine, Adriamycin,dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin orplatinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil,Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin,adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone),MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin,cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin),ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide, etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin, cyclophosphamide,etoposide, cytarabine, bleomycin, vincristine, methotrexate,leucovorin), VAD (vincristine, Adriamycin, dexamethasone), Regimen I(vincristine, Adriamycin, etoposide, cyclophosphamide) and VAPEC-B(vincristine, Adriamycin, prednisone, etoposide, cyclophosphamide,bleomycin).

The present invention does not intend to limit the type of cancer beingtreated to breast cancer. Cancers that may be treated using thecompositions and methods of the present invention include, for example,leukemia, carcinoma, lymphoma, astrocytoma, sarcoma, glioma,retinoblastoma, melanoma, Wilm's tumor, bladder cancer, colon cancer,hepatocellular cancer, pancreatic cancer, prostate cancer, lung cancer,liver cancer, stomach cancer, cervical cancer, testicular cancer, renalcell cancer, and brain cancer.

The present invention does not intend to limit the types of RNA used tosilence gene expression via RNA interference (RNAi). In one embodiment,the present invention contemplates the use of shRNAs, siRNAs, microRNAs(miRNAs), and single- or double-stranded analogues thereof, forsilencing gene expression.

The present invention does not intend to limit the compounds and/ormolecules used to silence gene expression to dsRNA molecules, such asshRNAs and siRNAs. In one embodiment, the present invention contemplatesthat inhibitors of cancer cells (e.g. breast cancer) may include smallmolecule inhibitors of hematopoietic cancers including, but not limitedto, ibrutinib (and analogues thereof).

In one embodiment, the instant invention provides a method of treatingcancer, comprising: a) providing a subject with cancer (e.g. breastcancer cells) and inhibitors of a gene encoding a cytoplasmic tyrosinekinase, and b) treating said subject with said inhibitor. In oneembodiment, the cytoplasmic tyrosine kinase is Bruton's Tyrosine Kinase.In one embodiment, the cytoplasmic tyrosine kinase is a variant ofBruton's Tyrosine Kinase comprising an amino-terminal extension. In oneembodiment, the extension comprises an additional 34 amino acids. In oneembodiment, the inhibitor is ibrutinib.

In one embodiment, the instant invention provides a method of treatingcancer, comprising: a) providing a subject with cancer (e.g. breastcancer cells) and inhibitors of a gene encoding the encoding anepidermal growth factor, and b) treating said subject with saidinhibitor. In one embodiment, the epidermal growth factor is HER2. Inone embodiment, the inhibitor is ibrutinib. In one embodiment, ibrutinibinhibits HER2 at a 10-fold lower concentration that lapatinib.

In another embodiment, the compounds of the present invention can becombined with other signal transduction inhibitors. Examples of suchagents include, by no way of limitation, antibody therapies such asHerceptin (trastuzumab), Erbitux (cetuximab), Yervoy (ipilimumab) andpertuzumab. Examples of such therapies also include, by no way oflimitation, small-molecule kinase inhibitors such as Imatinib (Gleevec),Sunitinib (Sutent), Sorafenib (Nexavar), Erlotinib (Tarceva), Gefitinib(Iressa), Dasatinib (Sprycel), Nilotinib (Tasigna), Lapatinib (Tykerb),Crizotinib (Xalkori), Ruxolitinib (Jakafi), Vemurafenib (Zelboraf),Vandetanib (Caprelsa), Pazopanib (Votrient), afatinib, alisertib,amuvatinib, axitinib, bosutinib, brivanib, canertinib, cabozantinib,cediranib, crenolanib, dabrafenib, dacomitinib, danusertib, dovitinib,foretinib, ganetespib, ibrutinib, iniparib, lenvatinib, linifanib,linsitinib, masitinib, momelotinib, motesanib, neratinib, niraparib,oprozomib, olaparib, pictilisib, ponatinib, quizartinib, regorafenib,rigosertib, rucaparib, saracatinib, saridegib, tandutinib, tasocitinib,telatinib, tivantinib, tivozanib, tofacitinib, trametinib, vatalanib,veliparib, vismodegib, volasertib, BMS-540215, BMS777607, JNJ38877605,TKI258, GDC-0941 (Folkes, et al., J. Med. Chem. 2008, 51, 5522), BZE235,and others.

In one embodiment, the instant invention provides a method of treatingcancer, comprising: a) providing a subject with cancer (e.g. breastcancer cells) and an inhibitor of a gene encoding the tyrosine kinaseLYN and b) treating said subject with said inhibitor. In one embodimentthe inhibitor is an shRNA. In another embodiment, the inhibitor is ansiRNA.

In one embodiment, the present invention contemplates a method oftreating cancer, comprising: a) providing: i) a subject with breastcancer cells, at least some of said breast cancer cells exhibitingresistance to a chemotherapeutic agent, and ii) an inhibitor of a geneencoding a cytoplasmic tyrosine kinase, and b) treating said subjectwith said inhibitor. In one embodiment, the cytoplasmic tyrosine kinaseis Bruton's Tyrosine Kinase. In one embodiment, the cytoplasmic tyrosinekinase is a variant of Bruton's Tyrosine Kinase comprising anamino-terminal extension. In one embodiment, the extension comprises anadditional 34 amino acids. In one embodiment the inhibitor comprisesinterfering double stranded RNA. In another embodiment, treating withthe inhibitor results in reduced proliferation of at least some of thebreast cancer cells within the subject. In one embodiment, the inhibitoris LFM-A13. In one embodiment, the inhibitor results in reducedproliferation of at least some breast cancer cells within the subjectidentified as resistant to the chemotherapeutic agent. In anotherembodiment, the chemotherapeutic agent is selected from the groupconsisting of AC (Adriamycin, cyclophosphamide), TAC (taxotere, AC),ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine), BEACOPP(bleomycin, etoposide, Adriamycin, cyclophosphamide, vincristine,procarbazine, prednisone), BEP (bleomycin, etoposide, platinum agent(cisplatin (Platinol)), CAF (cyclophosphamide, Adriamycin, fluorouracil(5-FU)), CAV (cyclophosphamide, Adriamycin, vincristine), CHOP(cyclophosphamide, Adriamycin, vincristine, prednisone), ChIVPP/EVA(chlorambucil, vincristine, procarbazine, prednisone, etoposide,vinblastine, Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine,Adriamycin, dexamethasone), DT-PACE (dexamethasone, thalidomide,cisplatin or platinol, Adriamycin, cyclophosphamide, etoposide), FAC(5-fluorouracil, Adriamycin, cyclophosphamide), m-BACOD (methotrexate,bleomycin, adriamycin, cyclophosphamide, Oncovin (vincristine),dexamethasone), MACOP-B (methotrexate, leucovorin (folinic acid),adriamycin, cyclophosphamide, Oncovin (vincristine), prednisone,bleomycin), ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide,etoposide+MOPP), ProMACE-CytaB OM (prednisone, Adriamycin,cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine,methotrexate, leucovorin), VAD (vincristine, Adriamycin, dexamethasone),Regimen I (vincristine, Adriamycin, etoposide, cyclophosphamide) andVAPEC-B (vincristine, Adriamycin, prednisone, etoposide,cyclophosphamide, bleomycin).

In one embodiment, the instant invention provides a method of treatingcancer, comprising: a) providing a subject with cancer (e.g. breastcancer cells) and inhibitors of a gene encoding the encoding acytoplasmic tyrosine kinase, and b) treating said subject with saidinhibitor. In one embodiment, the cytoplasmic tyrosine kinase isBruton's Tyrosine Kinase. In one embodiment, the cytoplasmic tyrosinekinase is a variant of Bruton's Tyrosine Kinase comprising anamino-terminal extension. In one embodiment, the extension comprises anadditional 34 amino acids. In one embodiment the inhibitors are shRNA.In one embodiment, the inhibtors are siRNA. In one embodiment, thepresent invention contemplates a combination of shRNA and siRNA. In oneembodiment, the shRNA targets an internal exon of BTK. In anotherembodiment, the siRNA targets BTK-C.

In one embodiment, the instant invention provides a method of treatingcancer, comprising: a) providing a subject with a cancer (e.g. breastcancer cells) with an acquired resistance to at least onechemotherapeutic agent, and inhibitors of a gene encoding the encoding acytoplasmic tyrosine kinase, and b) treating said subject with saidinhibitor. In one embodiment, the cytoplasmic tyrosine kinase isBruton's Tyrosine Kinase. In one embodiment, the cytoplasmic tyrosinekinase is a variant of Bruton's Tyrosine Kinase comprising anamino-terminal extension. In one embodiment, the extension comprises anadditional 34 amino acids. In one embodiment, the acquired resistance ofsaid cancer cells is to imatinib.

FIGURE LEGENDS

FIG. 1. An RNAi screen targeting tyrosine kinase genes in an ERBB2(HER2/neu) positive breast cancer. BT474 breast cancer cells weretransfected with 234 shRNA constructs targeting 83 protein tyrosinekinase genes. Three transfection mixes were produced for each shRNA andeach was transfected into triplicate wells of BT474 cells for 96 hours.AlamarBlue was used to monitor cell proliferation and viability. Theaverages of the nine parallel cultures were calculated for each shRNA,normalized to transfection efficiency, presented as % of the controlshRNA and sorted on the basis of effect. z-scores were calculated usingthe following formula: (normalized sample value−normalized data setmean)/data set standard deviation. shRNAs that produced z-scores lessthan −1.1 are presented in a list (Table 2).

FIG. 2. Btk knockdown in BT474 cells leads to increased apoptosis. (a)Brightfield image after 96 hr of siRNA knockdown of Btk in BT474 cells.(b-c) siRNA knockdown of Btk in BT474 cells (48 hr) results in increasedcleaved caspase-3 (CC3) compared to a scrambled siRNA. (b) Apoptoticcells were calculated as a percentage of the total cellular population.

FIG. 3. An alternative form of the Btk transcript is present in BT474breast cancer cells. (a) Nucleotide sequence 1-395 by from the publishedBtk sequence (accession # U13399) was aligned to the nucleotide sequenceobtained from BT474 cells using 5′RACE. Identical sequence ishighlighted in grey. The BT474 sequence obtained using 5′RACE translatesinto an additional 47 amino acid open reading frame (ORF) and containstwo additional methionine codons, highlighted in green, that are inframe with the methionine start codon of the published Btk gene(highlighted in green and with an arrow). (b) Schematic representationshowing the location of the Btk gene on the X-chromosome (b & c) andschematic representations comparing the location of the Btk-A and Btk-Cexon 1.

FIG. 4. BTK-C yields an 80 kD BTK specific product. (a) A schematicrepresentation showing the conserved domains of the BTK-A protein iscompared to a schematic of the predicted BTKC protein. (b) Total lysatefrom BT474 cells and a malignant B-cell line positive for Btk-A(Namalwa) were subjected to immunoblotting with the BTK antibody. (c)293FT cells were co-transfected with a BTK-A or BTK-C flag tagover-expression vector as well as a Btk shRNA or a control shRNAtargeting the firefly luciferase gene. (d) siRNAs targeting, stably,over-expressed BTK-C in BT474 cells leads to efficient knockdown. BT474cells were transfected with siRNAs and total lysate was used forimmunoblotting with the BTK antibody.

FIG. 5. BTK-C is activated in BT474 cells. (a) In BT474 cells both formsof the over-expressed Btk-C proteins are phosphorylated on tyrosineresidue 223, which becomes auto-phosphorylated after activation. Totallysate was prepared from BT474 cells containing the stably integratedBtk-A or Btk-C flag tag MarxIV vectors. Controls cells contain a stablyintegrated MarxIV flag tag vector encoding the beta-galactosidase gene(-gal) which retains its stop codon. Tyrosine phosphorylated BTK wasassessed by immunoprecipitation (IP) using anti-Flag and Western blot(WB) analysis using anti-BTK Phospho (pY223) and anti-BTK. (a) Thespecific BTK inhibitor LFM-A13 reduces phosphorylation of BTK. BT474cells containing the stably integrated Btk-A or Btk-C flag tag MarxIVvectors were incubated with 100 [μM LFM-A13 for 45 mins.Tyrosine-phosphorylated BTK was assessed by immunoprecipitation (IP)using anti-Flag and Western blot (WB) analysis using anti-BTK Phospho(pY223) and anti-BTK. (b & c) Inhibition of BTK auto-phosphorylationusing LFM-A13 results in increased apoptosis. BT474 cells incubated witheither 25 uM or 35 uM LFM-A13 results in increased cleaved caspase-3(CC3) compared to control cells treated with DMSO. (c) Apoptotic cellswere calculated as a percentage of the total cellular population.

FIG. 6. BTK protein is present in BT474 cellular cytoplasm. (a) Confocalimmunofluorescence images of BTK in BT474 cells. Left column: Alexa 568secondary ab (no primary ab); middle column. BTK ab; right column: FlagTag ab. (b) BT474 cells containing the stably integrated (left panels)control vector, (middle panels) BTK-A-flag vector, (right panels)BT-C-flag vector. Left panels: nuclei visualized with Hoechst; rightpanels: primary antibody (Btk or Flag) bound to secondary HRP conjugatedantibody tagged with Alexa 568 tag.

FIG. 7. Btk-C is more abundant in breast cancer cells than innon-tumorigenic breast cells or a malignant B-cell line. (a) qPCRprimers were designed to specifically target the Btk-C message and cDNAfrom the breast cancer cell lines BT474, MCF7, and MDA-MB-361, thenon-tumorigenic breast cell lines MCF10a and HMEC, as well as, amalignant B-cell line was amplified using SYBR Green. The breast cancercell lines BT474 and MCF7 had at least 4-fold more transcript comparedto the non-tumorigenic breast cell lines MCF10a and HMEC and themalignant B-cell line Namalwa. Fold change was calculated using thedelta, delta Ct method.

FIG. 8. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). tRNAwas isolated from the BT474 breast cancer cell line, a normal breastepithelial cell line (HMEC) and a Btk positive control cell line(Namalwa B-cells) and cDNA was amplified from the breast cancer cellsline BT474, a normal breast epithelial cell line (HMEC) and a positivecontrol cell line for BTK-A expression (B-cells) using three differentprimer pair combinations. Two distinct primer pairs were generated totarget the Btk transcript at different regions of the mRNA (5′UTR andBtk internal) and cDNA from each cell type was used as substrate in apolymerase chain reaction (PCR). (a) PCR products were amplified fromcDNAs isolated from B-cells for all primer pair combinations used. Aproduct was amplified from BT474 cDNA using the Btk internal forwardprimer but not when the 5′UTR forward primer was used. Differences inproduct size between BT474 and B cells in the rightmost panel are likelyan artifact of electrophoresis or may represent the presence of internalsplice variants in the Namalwa transcriptome. Data are representative ofthree replicated experiments.

FIG. 9. BTK-C but not BTK-A is expressed in breast cell lines. ReverseTranscriptase Polymerase Chain Reaction (RT-PCR). tRNA was isolated fromseveral breast cancer cell lines (BT474, MCF7, MDA-MB-361), and twonormal cell lines, HMEC and MCF10a and cDNA was amplified from each.Primer pairs were designed to specifically target the Btk-A or BTK-CmRNA sequence and cDNA from each cell type was used as substrate in apolymerase chain reaction (PCR). (a) A PCR product was amplified fromcDNA isolated from the B-cell line using the Btk-A specific primers butno product was amplified for any of the breast cell lines tested,whereas the BTK-C specific product was expressed preferentially in thebreast cancer cell lines. Data is representative of three replicatedexperiments.

FIG. 10. (a) siRNA knockdown of BTK in BT474 cells (48 hr) results inincreased cleaved caspase-3 (Caspase-3) staining indicative ofapoptosis. (b) Degree of apoptosis due to BTK knockdown in BT474 andMCF-7 cells was calculated as a percentage of the total cellularpopulation. The data were expressed as the mean of triplicate of thesamples transfected with the BTK siRNA relative to scrambled siRNAcontrol samples. Error bars represent standard deviation from theaverage of 3 replicates. Statistical significance between samples wascalculated using the student's t test, where (*) indicates a P value of<0.0001. FIG. 11. An alternative form of the BTK transcript is presentin BT474 breast cancer cells. (a) Nucleotide sequence 1-395 bp from thepublished BTK sequence (accession # U13399) was aligned to thenucleotide sequence obtained from BT474 cells using 5′RACE. Identicalsequence is highlighted in grey. The BT474 sequence obtained using5′RACE encodes an additional 34 amino acid open reading frame (ORF) andcontains two additional methionine codons, highlighted in green, thatare in frame with the methionine start codon from the published BTK gene(highlighted in green and with an arrow). (b) Schematic representationshowing alternate splicing of alternative first exons from both isoformsinferred from sequence analysis. Sequences are identical from exons 2through 19. (c) Map of BTK on the X-chromosome. BTKC exon 1transcription initiates divergently 255 by from the start site of theribosomal protein L36a gene.

FIG. 12. The BTK-C gene produces an 80 kD product. (a) A schematicrepresentation showing the domains of the BTK-A and predicted BTK-Cprotein. (b) Total lysate from breast lines and Namalwa B-cellssubjected to immunoblotting and probed with an anti-BTK antibody (BDTransduction Laboratory, 611116). (c) HEK293 cells co-transfected with aBTK-C flag vector and either BTK-C siRNAs or Non-Target siRNA. Totallysate was prepared 96 hrs post transfection and was used forimmunoblotting with anti-Flag antibody. (d) BT474 cells were transfectedwith two BTK-C specific siRNAs, non-target siRNA as a control andco-transfected with GFP to mark transfected cells. Transfected cellswere counted at 24 h and 96 h and the 96 hr to 24 hr ratio wascalculated and expressed as % of the control.

FIG. 13. BTK-C is activated in BT474 cells. In BT474 cells both forms ofthe over-expressed BTK-C proteins are phosphorylated on tyrosine residue223, which becomes auto-phosphorylated after activation. (a) BT474 cellscontaining the stably integrated BTK-A-flag, the BTK-C-flag or controlflag vector were treated with 100 μM LFM-A13 for 45 minsTyrosine-phosphorylated BTK was assessed by immunoprecipitation (IP)using anti-Flag (Stratagene) and immunoblot analysis using anti-BTKPhospho (pY223) and anti-BTK antibody (BTK-E9 Santa Cruz). (b)Inhibition of BTK auto-phosphorylation using LFM-A13 results inincreased apoptosis. BT474 cells incubated with 35 μM LFM-A13 for 48 hresults in increased cleaved caspase-3 (Caspase-3) compared to controlcells treated with DMSO. Apoptotic cells were calculated as a percentageof the total cellular population as in FIG. 10B. (c) BTK-C inhibitsapoptosis induced by Doxorubicin in MCF-10A cells. BTK-C expression invector control (10A-Vec) and MCF-10A-BTK-C (10A-BtkC) cells usinganti-Flag antibody. GAPDH is used as a loading control. (d) BTK-Cexpression reduces Doxorubicin-induced apoptosis as monitored by Cleavedcaspase-3 signal. 10A-Vec or 10A-BTK-C cells were either treated withDMSO (Con) or with 35 uM LFM-A13 for 24 hours, after that the cells werewashed with PBS for 3 times and added fresh medium with Doxorubicin (1uM) for 24 hours. Immunofluorescence was performed for cleavedcaspased-3 signal; cell nuclei were stained with Hoechst 33342. (e)Apoptotic cells were calculated as a percentage of the total cellularpopulation, as indicated B. Error bars indicate the standard deviationfrom three individual experiments, *P<0.01.

FIG. 14. BTK is more abundant in breast cancer cells compared tonon-tumorigenic breast cells. (a) BTK protein levels were examined innormal, matched breast tissues and breast carcinoma tissue in tissuemicroarrays using immunofluorescence microscopy. DAPI staining of nucleiis shown in cyan false color; anti-BTK (ProSci) staining is red. Tissuesamples and BTK classifications were (i) Normal-low level; (ii) benignhyperplasia-low level; (iii) Cancer-low-moderate/heterogenous; (iv)Cancer-heterogenous with strong positives; (v) Cancer-homogenousmoderate with nuclear; (vi) Cancer-negative. (b) BTK-C message is moreabundant than the BTK-A isoform in cancer cell lines. qPCR primersdesigned to specifically target the BTK-C message and cDNA from thebreast cancer cell lines BT474, MCF7, MDA-MB-361 and two non-tumorigenicbreast cell lines, HMEC and MCF 10a were amplified using SYBR Green.Fold change was calculated using the delta, delta Ct method. Error barsrepresent standard deviation from the average of 4 replicates.Statistical significance between samples was calculated using thestudent's t test, where (*) indicates a P value of <0.005 and (**)indicates a value of <0.0005. (c) BTK-C message is more abundant thanthe BTK-A isoform in breast tumors. cDNA prepared from RNA isolated fromhuman breast tissue was subjected to qPCR using primers specific forBTK-A and BTK-C isoforms. The same set of samples in another plate wasused for detection of actin mRNA. The data represent relative mRNAlevels of each BTK isoform normalized to actin.

FIG. 15. BTK-C promotes glucose uptake. (c) LFM-A13 inhibits glucoseuptake in 10A-BTK-C cells. 10A-Vec and 10A-BTK-C cells were eithertreated with DMSO (Control) or with 35 μM LFM-A13 for 24 hours, afterthat the cells were washed with PBS for 3 times and added 100 μm 2-NBDGfor 15 min. Immunofluorescence pictures were taken in INCELL-1000; (d)Fluorescence intensity was quantified in right. Error bars indicate thestandard deviation from three individual experiments, *P<0.01. (a) Theeffect of BTK-C on glucose uptake in breast cancer cell lines. MCF-7 andMDA-MB-361 cell lines were treated as in (c). (b) Fluorescence intensityshown at right panel, *p<0.01.

FIG. 16. BTK inhibition results in breast cancer cell death. Cell countsof MCF10A, MCF7 and BT474 cells treated with vehicle, 10 and 20 μmol/l.of the BTK kinase inhibitor PCI-32765 (ibrutinib) for 48 hours. Resultsare presented as percentage of control. Error bars indicate the standarddeviation for three individual experiments. MCF10A serves as a normal(i.e. non-cancerous cell) control. MCF7 and BT474 are cancer cell lines.

FIG. 17. The BTK-C gene produces an 80 kD product. (a) 293FT cellsco-transfected with a BTK-A or BTK-C flag over-expression vector andeither BTK shRNA or control shRNA. Total lysates were prepared 48 hourspost transfection and used for immunoblotting with anti-Flag antibody(Stratagene). (b) BT474 cells stably over-expressing BTK-C (MarxIV)transfected with siRNAs targeting BTK (48 hours) leads to efficient BTKknockdown compared to cells transfected with a control siRNA. Totallysates were used for immunoblotting with an anti-BTK antibody (ProSci).

FIG. 18. BTK is predominantly found in the cytoplasm of BT474 breastcancer cells. Confocal immunofluorescence images of BTK in BT474 cells.Left column: Alexa568 conjugated to secondary antibody (no primaryantibody); right column: anti-BTK antibody (ProSci); right column:anti-Flag antibody (Stratagene). Nuclei visualized with Hoechst;anti-BTK (ProSci) bound to secondary HRP conjugated antibody tagged withAlexa 568 tag.

FIG. 19. Ibrutinib (PCI-32765) is effective at lower concentrations thanLapatinib in killing SK-Br-3 Her2/neu positive breast cancer cells.SKBR3 cells were treated with (or without) 50 ng/ml EGF andconcomitantly exposed to different concentrations of Lapatinib. After 72hours, cells were fixed with 4% formaldehyde, stained with Hoechst andcell number determined.

FIG. 20. PCI-32765 blocks ERRB2 (HER2/neu) activation in breast cancercells (red arrows). EGF treatment has been shown to activate apro-survival pathway whose reactivation correlates with EGF-stimulatedERK activation in tyrosine kinase inhibitor treated cells. EGFcounteracts lapatinib's effect, causing ERK re-activation whichcorrelates with potential decreased efficacy and drug resistance (greenarrows). EGF treatment does not bypass the effects of PCI-32765 oninhibiting ERK activation (orange arrows) and prevents ERK reactivationin the presence of lapatinib (blue arrow). SKbr3 cells were treated withlapatinib (1 μM) with EGF (50 ng/ml), PCI-32765 (1 μM) or PCI-32765 (1μM) with EGF (50 ng/ml). After 3 hours, cells were lysed. Immunoblotsshowing effect of kinase inhibition with or without EGF on HER2, AKT andERK phosphorylation. Anti-pHER2 (1221), pAKT (437) and pERK (202/204).

FIG. 21. Sequence alignment. The ability of PCI-32765 to block ERRB2(HER2/neu) activation in breast cancer cells may be due to similaritieswith the BTK active site. (a) EGFR family members EGFR, ERBB2 and ERRB4share the PCI-32765-targeted cysteine residue found in BTK (red box).(b) Several other non-TEC family kinases do not share thePCI-32765-targeted cysteine residue (red box).

FIG. 22. EGF treatment counteracts lapatinib to a much larger degreethan PCI-32765. SKBR3 cells were treated with (or without) 50 ng/ml EGFand concomitantly exposed to different concentrations of lapatinib orPCI-32765. After 72 hours, cell number was determined.

DEFINITIONS

To facilitate the understanding of this invention a number of terms (setoff in quotation marks in this Definitions section) are defined below.Terms defined herein (unless otherwise specified) have meanings ascommonly understood by a person of ordinary skill in the areas relevantto the present invention. As used in this specification and its appendedclaims, terms such as “a”, “an” and “the” are not intended to refer toonly a singular entity, but include the general class of which aspecific example may be used for illustration, unless the contextdictates otherwise. The terminology herein is used to describe specificembodiments of the invention, but their usage does not delimit theinvention, except as outlined in the claims.

The phrase “chosen from A, B, and C” as used herein, means selecting oneor more of A, B, C.

As used herein, absent an express indication to the contrary, the term“or” when used in the expression “A or B,” where A and B refer to acomposition, disease, product, etc., means one or the other, or both. Asused herein, the term “comprising” when placed before the recitation ofsteps in a method means that the method encompasses one or more stepsthat are additional to those expressly recited, and that the additionalone or more steps may be performed before, between, and/or after therecited steps. For example, a method comprising steps a, b, and cencompasses a method of steps a, b, x, and c, a method of steps a, b, c,and x, as well as a method of steps x, a, b, and c. Furthermore, theterm “comprising” when placed before the recitation of steps in a methoddoes not (although it may) require sequential performance of the listedsteps, unless the context clearly dictates otherwise. For example, amethod comprising steps a, b, and c encompasses, for example, a methodof performing steps in the order of steps a, c, and b, the order ofsteps c, b, and a, and the order of steps c, a, and b, etc.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weights, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersin the specification and claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and without limiting theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parametersdescribing the broad scope of the invention are approximations, thenumerical values in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains standarddeviations that necessarily result from the errors found in thenumerical value's testing measurements.

The term “not” when preceding, and made in reference to, anyparticularly named molecule (mRNA, etc.) or phenomenon (such asbiological activity, biochemical activity, etc.) means that only theparticularly named molecule or phenomenon is excluded.

The term “altering” and grammatical equivalents as used herein inreference to the level of any substance and/or phenomenon refers to anincrease and/or decrease in the quantity of the substance and/orphenomenon, regardless of whether the quantity is determinedobjectively, and/or subjectively.

The terms “increase,” “elevate,” “raise,” and grammatical equivalentswhen used in reference to the level of a substance and/or phenomenon ina first sample relative to a second sample, mean that the quantity ofthe substance and/or phenomenon in the first sample is higher than inthe second sample by any amount that is statistically significant usingany art-accepted statistical method of analysis. In one embodiment, theincrease may be determined subjectively, for example when a patientrefers to their subjective perception of disease symptoms, such as pain,clarity of vision, etc. In another embodiment, the quantity of thesubstance and/or phenomenon in the first sample is at least 10% greaterthan the quantity of the same substance and/or phenomenon in a secondsample. In another embodiment, the quantity of the substance and/orphenomenon in the first sample is at least 25% greater than the quantityof the same substance and/or phenomenon in a second sample. In yetanother embodiment, the quantity of the substance and/or phenomenon inthe first sample is at least 50% greater than the quantity of the samesubstance and/or phenomenon in a second sample. In a further embodiment,the quantity of the substance and/or phenomenon in the first sample isat least 75% greater than the quantity of the same substance and/orphenomenon in a second sample. In yet another embodiment, the quantityof the substance and/or phenomenon in the first sample is at least 90%greater than the quantity of the same substance and/or phenomenon in asecond sample. Alternatively, a difference may be expressed as an“n-fold” difference.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” andgrammatical equivalents when used in reference to the level of asubstance and/or phenomenon in a first sample relative to a secondsample, mean that the quantity of substance and/or phenomenon in thefirst sample is lower than in the second sample by any amount that isstatistically significant using any art-accepted statistical method ofanalysis. In one embodiment, the reduction may be determinedsubjectively, for example when a patient refers to their subjectiveperception of disease symptoms, such as pain, clarity of vision, etc. Inanother embodiment, the quantity of substance and/or phenomenon in thefirst sample is at least 10% lower than the quantity of the samesubstance and/or phenomenon in a second sample. In another embodiment,the quantity of the substance and/or phenomenon in the first sample isat least 25% lower than the quantity of the same substance and/orphenomenon in a second sample. In yet another embodiment, the quantityof the substance and/or phenomenon in the first sample is at least 50%lower than the quantity of the same substance and/or phenomenon in asecond sample. In a further embodiment, the quantity of the substanceand/or phenomenon in the first sample is at least 75% lower than thequantity of the same substance and/or phenomenon in a second sample. Inyet another embodiment, the quantity of the substance and/or phenomenonin the first sample is at least 90% lower than the quantity of the samesubstance and/or phenomenon in a second sample. Alternatively, adifference may be expressed as an “n-fold” difference.

A number of terms herein relate to cancer. “Cancer” is intended hereinto encompass all forms of abnormal or improperly regulated reproductionof cells in a subject. “Subject” and “patient” are used hereininterchangeably, and a subject may be any mammal but is preferably ahuman. A “reference subject” herein refers to an individual who does nothave cancer. The “reference subject” thereby provides a basis to whichanother cell (for example a cancer cell) can be compared.

The growth of cancer cells (“growth” herein referring generally to celldivision but also to the growth in size of masses of cells) ischaracteristically uncontrolled or inadequately controlled, as is thedeath (“apoptosis”) of such cells. Local accumulations of such cellsresult in a tumor. More broadly, and still denoting “tumors” herein areaccumulations ranging from a cluster of lymphocytes at a site ofinfection to vascularized overgrowths, both benign and malignant. A“malignant” tumor (as opposed to a “benign” tumor) herein comprisescells that tend to migrate to nearby tissues, including cells that maytravel through the circulatory system to invade or colonize tissues ororgans at considerable remove from their site of origin in the “primarytumor,” so-called herein. Metastatic cells are adapted to penetrateblood vessel wells to enter (“intravasate”) and exit (“extravasate”)blood vessels. Tumors capable of releasing such cells are also referredto herein as “metastatic.” The term is used herein also to denote anycell in such a tumor that is capable of such travel, or that is enroute, or that has established a foothold in a target tissue. Forexample, a metastatic breast cancer cell that has taken root in the lungis referred to herein as a “lung metastasis.” Metastatic cells may beidentified herein by their respective sites of origin and destination,such as “breast-to-bone metastatic.” In the target tissue, a colony ofmetastatic cells can grow into a “secondary tumor,” so called herein.Primary tumors are thought to derive from a benign or normal cellthrough a process referred to herein as “cancer progression.” Accordingto this view, the transformation of a normal cell to a cancer cellrequires changes (usually many of them) in the cell's biochemistry. Thechanges are reflected clinically as the disease progresses throughstages. Even if a tumor is “clonogenic” (as used herein, an accumulationof the direct descendants of a parent cell), the biochemistry of theaccumulating cells changes in successive generations, both because theexpression of the genes (controlled by so-called “epigenetic” systems)of these cells becomes unstable and because the genomes themselveschange. In normal somatic cells, the genome (that is, all the genes ofan individual) is stored in the chromosomes of each cell (setting asidethe mitochondrial genome). The number of copies of any particular geneis largely invariant from cell to cell. By contrast, “genomicinstability” is characteristic of cancer progression. A genome in acancer cell can gain (“genomic gain”) or lose (“genomic loss”) genes,typically because an extra copy of an entire chromosome appears(“trisomy”) or a region of a chromosome replicates itself (“genomicgain” or, in some cases, “genomic amplification”) or drops out when thecell divides. Thus, the “copy number” of a gene or a set of genes,largely invariant among normal cells, is likely to change in cancercells (referred to herein as a “genomic event”), which affects the totalexpression of the gene or gene set and the biological behavior(“phenotype”) of descendent cells. Thus, in cancer cells, “geneactivity” herein is determined not only by the multiple “layers” ofepigenetic control systems and signals that call forth expression of thegene but by the number of times that gene appears in the genome. Theterm “epigenetic” herein refers to any process in an individual that, inoperation, affects the expression of a gene or a set of genes in thatindividual, and stands in contrast to the “genetic” processes thatgovern the inheritance of genes in successive generations of cells orindividuals.

Certain regions of chromosomes, depending upon the specific type ofcancer, have proven to be hot spots for genomic gain inasmuch asincreases in copy number in the genomes of cells from multiple donorstend to occur in one or a few specific regions of a specific chromosome.Such hot spots are referred to herein as sites of “recurrent genomicgain.” The term is to be distinguished from “recurrent cancer,” whichrefers to types of cancer that are likely to recur after an initialcourse of therapy, resulting in a “relapse.” A number of terms hereinrelate to methods that enable the practitioner to examine many distinctgenes at once. By these methods, sets of genes (“gene sets”) have beenidentified wherein each set has biologically relevant and distinctiveproperties as a set. Devices (which may be referred to herein as“platforms”) in which each gene in a significant part of an entiregenome is isolated and arranged in an array of spots, each spot havingits own “address,” enable one to detect, quantitatively, many thousandsof the genes in a cell. More precisely, these “microarrays” typicallydetect expressed genes (an “expressed” gene is one that is activelytransmitting its unique biochemical signal to the cell in which the generesides). Microarray data, inasmuch as they display the expression ofmany genes at once, permit the practitioner to view “gene expressionprofiles” in a cell and to compare those profiles cell-to-cell toperform so-called “comparative analyses of expression profiles.” Suchmicroarray-based “expression data” are capable of identifying genes thatare “over-expressed” (or under-expressed) in, for example, a diseasecondition. An over-expressed gene may be referred to herein as having ahigh “expression score.”

The aforementioned methods for examining gene sets employ a number ofwell-known methods in molecular biology, to which references are madeherein. A gene is a heritable chemical code resident in, for example, acell, virus, or bacteriophage that an organism reads (decodes, decrypts,transcribes) as a template for ordering the structures of biomoleculesthat an organism synthesizes to impart regulated function to theorganism. Chemically, a gene is a heteropolymer comprised of subunits(“nucleotides”) arranged in a specific sequence. In cells, suchheteropolymers are deoxynucleic acids (“DNA”) or ribonucleic acids(“RNA”). DNA forms long strands. Characteristically, these strands occurin pairs. The first member of a pair is not identical in nucleotidesequence to the second strand, but complementary. The tendency of afirst strand to bind in this way to a complementary second strand (thetwo strands are said to “anneal” or “hybridize”), together with thetendency of individual nucleotides to line up against a single strand ina complementarily ordered manner accounts for the replication of DNA.

Experimentally, nucleotide sequences selected for their complementaritycan be made to anneal to a strand of DNA containing one or more genes. Asingle such sequence can be employed to identify the presence of aparticular gene by attaching itself to the gene. This so called “probe”sequence is adapted to carry with it a “marker” that the investigatorcan readily detect as evidence that the probe struck a target. As usedherein, the term “marker” relates to any surrogate the artisan may useto “observe” an event or condition that is difficult or impossible todetect directly. In some contexts herein, the marker is said to “target”the condition or event. In other contexts, the condition or event isreferred to as the target for the marker. Sequences used as probes maybe quite small (e.g., “oligonucleotides” of <20 nucleotides) or quitelarge (e.g., a sequence of 100,000 nucleotides in DNA from a “bacterialartificial chromosome” or “BAC”). A BAC is a bacterial chromosome (or aportion thereof) with a “foreign” (typically, human) DNA fragmentinserted in it. BACs are employed in a technique referred to herein as“fluorescence in situ hybridization” or “FISH.” A BAC or a portion of aBAC is constructed that has (1) a sequence complementary to a region ofinterest on a chromosome and (2) a marker whose presence is discernibleby fluorescence. The chromosomes of a cell or a tissue are isolated (ona glass slide, for example) and treated with the BAC construct. Excessconstruct is washed away and the chromosomes examined microscopically tofind chromosomes or, more particularly, identifiable regions ofchromosomes that fluoresce.

Alternatively, such sequences can be delivered in pairs selected tohybridize with two specific sequences that bracket a gene sequence. Acomplementary strand of DNA then forms between the “primer pair.” In onewell-known method, the “polymerase chain reaction” or “PCR,” theformation of complementary strands can be made to occur repeatedly in anexponential amplification. A specific nucleotide sequence so amplifiedis referred to herein as the “amplicon” of that sequence. “QuantitativePCR” or “qPCR” herein refers to a version of the method that allows theartisan not only to detect the presence of a specific nucleic acidsequence but also to quantify how many copies of the sequence arepresent in a sample, at least relative to a control. As used herein,“qRTPCR” may refer to “quantitative real-time PCR,” used interchangeablywith “qPCR” as a technique for quantifying the amount of a specific DNAsequence in a sample. However, if the context so admits, the sameabbreviation may refer to “quantitative reverse transcriptase PCR,” amethod for determining the amount of messenger RNA present in a sample.Since the presence of a particular messenger RNA in a cell indicatesthat a specific gene is currently active (being expressed) in the cell,this quantitative technique finds use, for example, in gauging the levelof expression of a gene.

Collectively, the genes of an organism constitute its genome. The term“genomic DNA” may refer herein to the entirety of an organism's DNA orto the entirety of the nucleotides comprising a single gene in anorganism. A gene typically contains sequences of nucleotides devoted tocoding (“exons”), and non-coding sequences that contribute in one way oranother to the decoding process (“introns”).

The term “gene” refers to a nucleic acid (e.g., DNA) comprisingcovalently linked nucleotide monomers arranged in a particular sequencethat comprises a coding sequence necessary for the production of apolypeptide or precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). Thepolypeptide can be encoded by a full-length coding sequence or by anyportion of the coding sequence so long as the desired activities orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region together with the sequenceslocated adjacent to the coding region on both the 5′ and 3′ ends, suchthat the gene corresponds to the length of the full-length mRNA (alsoreferred to as “pre-mRNA,” “nuclear RNA,” or “primary transcript RNA”)transcribed from it. The sequences that are located 5′ of the codingregion and are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA (thecoding region(s) only) and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

Encoding in DNA (and messenger RNA) is accomplished by 3-memberednucleotide sequences called “codons.” Each codon encrypts an amino acid,and the sequence of codons encrypts the sequence of amino acids thatidentifies a particular protein. The code for a given gene is embeddedin a (usually) much longer nucleotide sequence and is distinguishable tothe cell's decoding system from the longer sequence by a “start codon”and a “stop” codon. The decoding system reads the sequence framed bythese two codons (the so-called “open reading frame”). The readable codeis transcribed into messenger RNA which itself comprises sites thatensure coherent translation of the code from nucleic acid to protein. Inparticular, the open reading frame is delimited by a so-called“translation initiation” codon and “translation termination” codon.

The term “plasmid” as used herein, refers to a small, independentlyreplicating, piece of DNA Similarly, the term “naked plasmid” refers toplasmid DNA devoid of extraneous material typically used to effecttransfection. As used herein, a “naked plasmid” refers to a plasmidsubstantially free of calcium-phosphate, DEAE-dextran, liposomes, and/orpolyamines. As used herein, the term “purified” refers to molecules(polynucleotides or polypeptides) that are removed from their naturalenvironment, isolated or separated. “Purified” molecules are at least50% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated.

The term “recombinant DNA” refers to a DNA molecule that is comprised ofsegments of DNA joined together by means of molecular biologytechniques. Similarly, the term “recombinant protein” refers to aprotein molecule that is expressed from recombinant DNA.

The term “fusion protein” as used herein refers to a protein formed byexpression of a hybrid gene made by combining two gene sequences.Typically this is accomplished by cloning a cDNA into an expressionvector in frame (i.e., in an arrangement that the cell can transcribe asa single mRNA molecule) with an existing gene. The fusion partner mayact as a reporter (e.g., (βgal) or may provide a tool for isolationpurposes (e.g., GST).

Where an amino acid sequence is recited herein to refer to an amino acidsequence of a protein molecule, “amino acid sequence” and like terms,such as “polypeptide” or “protein” are not meant to limit the amino acidsequence to the complete, native amino acid sequence associated with therecited protein molecule. Rather the terms “amino acid sequence” and“protein” encompass partial sequences, and modified sequences.

The term “wild type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild type gene is the variant mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene.

In contrast, the terms “modified,” “mutant,” and “variant” (when thecontext so admits) refer to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Insome embodiments, the modification comprises at least one nucleotideinsertion, deletion, or substitution.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to reduction in binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous sequence to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target. When used in reference to a single-strandednucleic acid sequence, the term “substantially homologous” refers to anyprobe that can hybridize (i.e., it is the complement of) thesingle-stranded nucleic acid sequence under conditions of low stringencyas described above.

As used herein, the term “competes for binding” when used in referenceto a first and a second polypeptide means that the first polypeptidewith an activity binds to the same substrate as does the secondpolypeptide with an activity. In one embodiment, the second polypeptideis a variant of the first polypeptide (e.g., encoded by a differentallele) or a related (e.g., encoded by a homolog) or dissimilar (e.g.,encoded by a second gene having no apparent relationship to the firstgene) polypeptide. The efficiency (e.g., kinetics or thermodynamics) ofbinding by the first polypeptide may be the same as or greater than orless than the efficiency of substrate binding by the second polypeptide.For example, the equilibrium binding constant (K_(D)) for binding to thesubstrate may be different for the two polypeptides.

As used herein, the term “hybridization” refers to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is impacted by such factors as the degree of complementaritybetween the nucleic acids, stringency of the conditions involved, theT_(m) of the formed hybrid, and the G:C ratio within the nucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with 85-100% identity, preferably 70-100%identity). With medium stringency conditions, nucleic acid base pairingwill occur between nucleic acids with an intermediate frequency ofcomplementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with 50-70% identity).Thus, conditions of “weak” or “low” stringency are often required withnucleic acids that are derived from organisms that are geneticallydiverse, as the frequency of complementary sequences is usually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 100 to about 1000 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 100 to about 1000 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 gFicoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42° C. when a probe of about 100 to about 1000nucleotides in length is employed.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 85% to 95%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with the other nucleicacid sequences that have from 85% to 95% homology to the first nucleicacid sequence.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (Smithand Waterman, Adv. Appl. Math., 2: 482, 1981) by the homology alignmentalgorithm of Needleman and Wunsch (Needleman and Wunsch, I Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman(Pearson and Lipman, Proc. Natl. Acad. Sci., U.S.A., 85:2444, 1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected. Theterm “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 85 percentsequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence, for example, as a segment of thefull-length sequences of the compositions claimed in the presentinvention.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having acidic side chains is glutamic acid and asparticacid; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

“Amplification” is used herein in two different ways. A given genetypically appears in a genome once, on one chromosome. Since chromosomesin somatic cells of eukaryotes are in general paired, two copies oralleles of each gene are found. In some conditions, such as cancer,replication of chromosome pairs during cell division is disturbed sothat multiple copies of a gene or chromosome accrue over successivegenerations. The phenomenon is referred to generally (and herein) as“amplification.”

In the context of molecular biological experimentation, the term is useddifferently. Experimentally, “amplification” is used in relation to aspecial case of nucleic acid replication involving template specificity.It is to be contrasted with non-specific template replication (i.e.,replication that is template-dependent but not dependent on a specifictemplate). Template specificity is here distinguished from fidelity ofreplication (i.e., synthesis of the proper polynucleotide sequence) andnucleotide (ribo- or deoxyribo-) specificity. Template specificity isfrequently described in terms of “target” specificity. Target sequencesare “targets” in the sense that they are sought to be sorted out fromother nucleic acid Amplification techniques have been designed primarilyfor this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, under theconditions in which they are used, will process only specific sequencesof nucleic acids in a heterogeneous mixture of nucleic acids. Inparticular, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences.

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particularsequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of Mullis (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, hereby incorporated by reference), that describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.”

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding gene includes, by way of example, suchnucleic acid in cells ordinarily expressing gene where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may single-stranded), but may containboth the sense and anti-sense strands (i.e., the oligonucleotide orpolynucleotide may be double-stranded).

The terms “fragment” and “portion” when used in reference to anucleotide sequence (as in “a portion of a given nucleotide sequence”)refers to partial segments of that sequence. The fragments may range insize from four nucleotides to the entire nucleotide sequence minus onenucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).

Similarly, the terms “fragment” and “portion” when used in reference toa polypeptide sequence refers to partial segments of that sequence. Insome embodiments, the portion has an amino-terminal and/orcarboxy-terminal deletion as compared to the native protein, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the amino acid sequence deduced from a full-length cDNAsequence. Fragments are preferably at least 4 amino acids long, morepreferably at least 50 amino acids long, and most preferably at least 50amino acids long or longer (the entire amino acid sequence minus onamino acid). In particularly preferred embodiments, the portioncomprises the amino acid residues required for intermolecular binding ofthe compositions of the present invention with its various ligandsand/or substrates.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid.

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques. Similarly, the term “recombinantprotein” or “recombinant polypeptide” as used herein refers to a proteinmolecule that is expressed from a recombinant DNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences, that arethe native protein contains only those amino acids found in the proteinas it occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp. 9.31-9.58, 1989).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (Sambrook, et al., supra, pp. 7.39-7.52,1989).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabelled antibodies

As used herein, the term “transgenic” refers to a cell or organism whosegenome has been heritably altered by genetically engineering into thegenome a gene (“transgene”) not normally part of it or removing from ita gene ordinarily present (a “knockout” gene). The “transgene” or“foreign gene” may be placed into an organism by introducing it intonewly fertilized eggs or early embryos. The term “foreign gene” refersto any nucleic acid (e.g., gene sequence) that is introduced into thegenome of an animal by experimental manipulations and may include genesequences found in that animal so long as the introduced gene does notreside in the same location as does the naturally-occurring gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term host cell refers to any eukaryotic orprokaryotic cell (e.g. bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell in the sense thatthe foreign DNA will be passed on to daughter cells. The termencompasses transfections of foreign DNA into the cytoplasm only. Ingeneral, however, the foreign DNA reaches the nucleus of the transfectedcell and persists there for several days. During this time the foreignDNA is subject to the regulatory controls that govern the expression ofendogenous genes in the chromosomes. The term “transient transfectant”refers to cells that have taken up foreign DNA but have failed tointegrate this DNA. The term “transient transfection” encompassestransfection of foreign DNA into the cytoplasm only

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofis modified to optimize conditions for particular types of cells. Theart is well aware of these numerous modifications.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. Such compositions may be employed ashybridization probes, typically in an aqueous solution containing salts(e.g., NaCl), detergents (e.g., SDS), and other components (e.g.,Denhardt's solution, dry milk, salmon sperm DNA, etc.).

The terms “N-terminus” “NH₂-terminus” and “amino-terminus” refer to theamino acid residue corresponding to the methionine encoded by the startcodon (e.g., position or residue 1). In contrast the terms “C-terminus”“COOH-terminus” and “carboxy terminus” refer to the amino acid residueencoded by the final codon (e.g., last or final residue prior to thestop codon).

The term “conservative substitution” as used herein refers to a changethat takes place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids can be divided intofour families: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine). Whether a change in theamino acid sequence of a peptide results in a functional homolog can bereadily determined by assessing the ability of the variant peptide tofunction in a fashion similar to the wild-type protein. Peptides havingmore than one replacement can readily be tested in the same manner. Incontrast, the term “non-conservative substitution” refers to a change inwhich an amino acid from one family is replaced with an amino acid fromanother family (e.g., replacement of a glycine with a tryptophan).Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological activity can be foundusing computer programs (e.g., LASERGENE software, DNASTAR Inc.,Madison, Wis.

A peptide sequence and nucleotide sequence may be “endogenous” or“heterologous” (i.e., “foreign”). The term “endogenous” refers to asequence which is naturally found in the cell or virus into which it isintroduced so long as it does not contain some modification relative tothe naturally-occurring sequence. The term “heterologous” refers to asequence which is not endogenous to the cell or virus into which it isintroduced. For example, heterologous DNA includes a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA alsoincludes a nucleotide sequence which is naturally found in the cell orvirus into which it is introduced and which contains some modificationrelative to the naturally-occurring sequence. Generally, although notnecessarily, heterologous DNA encodes heterologous RNA and heterologousproteins that are not normally produced by the cell or virus into whichit is introduced. Examples of heterologous DNA include reporter genes,transcriptional and translational regulatory sequences, DNA sequenceswhich encode selectable marker proteins (e.g., proteins which conferdrug resistance), etc. In preferred embodiments, the terms “heterologousantigen” and “heterologous sequence” refer to a non-hepadna virusantigen or amino acid sequence including but not limited to microbialantigens, mammalian antigens and allergen antigens.

The terms “peptide,” “peptide sequence,” “amino acid sequence,”“polypeptide,” and “polypeptide sequence” are used interchangeablyherein to refer to at least two amino acids or amino acid analogs whichare covalently linked by a peptide bond or an analog of a peptide bond.The term peptide includes oligomers and polymers of amino acids or aminoacid analogs. The term peptide also includes molecules which arecommonly referred to as peptides, which generally contain from about two(2) to about twenty (20) amino acids. The term peptide also includesmolecules which are commonly referred to as polypeptides, whichgenerally contain from about twenty (20) to about fifty amino acids(50). The term peptide also includes molecules which are commonlyreferred to as proteins, which generally contain from about fifty (50)to about three thousand (3000) amino acids. The amino acids of thepeptide may be L-amino acids or D-amino acids. A peptide, polypeptide orprotein may be synthetic, recombinant or naturally occurring. Asynthetic peptide is a peptide which is produced by artificial means invitro

The terms “oligosaccharide” and “OS” antigen refer to a carbohydratecomprising up to ten component sugars, either O or N linked to the nextsugar. Likewise, the terms “polysaccharide” and “PS” antigen refer topolymers of more than ten monosaccharide residues linked glycosidicallyin branched or unbranched chains

As used herein, the term “mammalian sequence” refers to synthetic,recombinant or purified sequences (preferably sequence fragmentscomprising at least one B cell epitope) of a mammal. Exemplary mammaliansequences include cytokine sequence, MHC class I heavy chain sequences,MHC class II alpha and beta chain sequences, and amyloid β-peptidesequences.

The terms “mammals” and “mammalian” refer animals of the class mammaliawhich nourish their young by fluid secreted from mammary glands of themother, including human beings. The class “mammalian” includes placentalanimals, marsupial animals, and monotrematal animals. An exemplary“mammal” may be a rodent, primate (including simian and human) ovine,bovine, ruminant, lagomorph, porcine, caprine, equine, canine, feline,ave, etc. Preferred non-human animals are selected from the orderRodentia.

Preferred embodiments of the present invention are primarily directed tovertebrate (backbone or notochord) members of the animal kingdom.

The terms “patient” and “subject” refer to a mammal that may be treatedusing the methods of the present invention.

The term “control” refers to subjects or samples which provide a basisfor comparison for experimental subjects or samples. For instance, theuse of control subjects or samples permits determinations to be maderegarding the efficacy of experimental procedures. In some embodiments,the term “control subject” refers to a subject that which receives amock treatment (e.g., saline alone).

The terms “diluent” and “diluting agent” as used herein refer to agentsused to diminish the strength of an admixture. Exemplary diluentsinclude water, physiological saline solution, human serum albumin, oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents, antibacterial agents such as benzyl alcohol, antioxidants suchas ascorbic acid or sodium bisulphite, chelating agents such as ethylenediamine-tetra-acetic acid, buffers such as acetates, citrates orphosphates and agents for adjusting the osmolarity, such as sodiumchloride or dextrose.

The terms “carrier” and “vehicle” as used herein refer to usuallyinactive accessory substances into which a pharmaceutical substance issuspended. Exemplary carriers include liquid carriers (such as water,saline, culture medium, saline, aqueous dextrose, and glycols) and solidcarriers (such as carbohydrates exemplified by starch, glucose, lactose,sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid andglutathione, and hydrolyzed proteins.

The term “derived” when in reference to a peptide derived from a source(such as a microbe, cell, etc.) as used herein is intended to refer to apeptide which has been obtained (e.g., isolated, purified, etc.) fromthe source. Alternatively, or in addition, the peptide may begenetically engineered and/or chemically synthesized.

The terms “operably linked,” “in operable combination” and “in operableorder” as used herein refer to the linkage of nucleic acid sequencessuch that they perform their intended function. For example, operablylinking a promoter sequence to a nucleotide sequence of interest refersto linking the promoter sequence and the nucleotide sequence of interestin a manner such that the promoter sequence is capable of directing thetranscription of the nucleotide sequence of interest and/or thesynthesis of a polypeptide encoded by the nucleotide sequence ofinterest. Similarly, operably linking a nucleic acid sequence encoding aprotein of interest means linking the nucleic acid sequence toregulatory and other sequences in a manner such that the protein ofinterest is expressed. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The terms “C-terminal portion,” “COOH-terminal portion,” “carboxyterminal portion,” “C-terminal domain,” “COOH-terminal domain,” and“carboxy terminal domain,” when used in reference to an amino acidsequence of interest refer to the amino acid sequence (and portionsthereof that is located from approximately the middle of the amino acidsequence of interest to the C-terminal-most amino acid residue of thesequence of interest. The terms “specific binding,” “bindingspecificity,” and grammatical equivalents thereof when made in referenceto the binding of a first molecule (such as a polypeptide, glycoprotein,nucleic acid sequence, etc.) to a second molecule (such as apolypeptide, glycoprotein, nucleic acid sequence, etc.) refer to thepreferential interaction between the first molecule with the secondmolecule as compared to the interaction between the second molecule witha third molecule. Specific binding is a relative term that does notrequire absolute specificity of binding; in other words, the term“specific binding” does not require that the second molecule interactwith the first molecule in the absence of an interaction between thesecond molecule and the third molecule. Rather, it is sufficient thatthe level of interaction between the first molecule and the secondmolecule is higher than the level of interaction between the secondmolecule with the third molecule. “Specific binding” of a first moleculewith a second molecule also means that the interaction between the firstmolecule and the second molecule is dependent upon the presence of aparticular structure on or within the first molecule; in other words thesecond molecule is recognizing and binding to a specific structure on orwithin the first molecule rather than to nucleic acids or to moleculesin general. For example, if a second molecule is specific for structure“A” that is on or within a first molecule, the presence of a thirdnucleic acid sequence containing structure A will reduce the amount ofthe second molecule which is bound to the first molecule.

For example, the term “has the biological activity of a specificallynamed protein” when made in reference to the biological activity of avariant of the specifically named protein refers, for example, to aquantity of binding of an antibody that is specific for the specificallynamed protein to the variant which is preferably greater than 50%(preferably from 50% to 500%, more preferably from 50% to 200%, mostpreferably from 50% to 100%), as compared to the quantity of binding ofthe same antibody to the specifically named protein.

Reference herein to any specifically named nucleotide sequence includeswithin its scope fragments, homologs, and sequences that hybridize understringent condition to the specifically named nucleotide sequence. Theterm “homolog” of a specifically named nucleotide sequence refers to anoligonucleotide sequence which exhibits greater than or equal to 50%identity to the sequence of interest. Alternatively, or in addition, ahomolog of any specifically named nucleotide sequence is defined as anoligonucleotide sequence which has at least 95% identity with thesequence of the nucleotide sequence in issue. In another embodiment, thesequence of the homolog has at least 90% identity, and preferably atleast 85% identity with the sequence of the nucleotide sequence inissue.

Exons, introns, genes and entire gene-sets are characteristicallylocatable with respect to one another. That is, they have generallyinvariant “genomic loci” or “genomic positions.” Genes distributedacross one or several chromosomes can be mapped to specific locations onspecific chromosomes. The field of “cytogenetics” addresses severalaspects of gene mapping. First, optical microscopy reveals features ofchromosomes that are useful as addresses for genes. In humans,chromosomes are morphologically distinguishable from one another andeach (except for the Y-chromosome) has two distinct arms separated by a“centromere.” Each arm has distinctive “bands” occupied by specificgenes. Disease-related changes in chromosome number and changes inbanding form the basis for diagnosing a number of diseases.“Microdissection” of chromosomes and DNA analysis of the microdissectedfragments have connected specific DNA sequences to specific locations onchromosomes. In cancer, a region of a chromosome may duplicate oramplify itself or drop out entirely. FISH, mentioned above, and“comparative genomic hybridization” (“CGH”) have extended the reach ofcytogenetic analysis to the extent of measuring genome alterationswithin and between individuals. CGH, for example, in which chromosomesfrom a normal cell are hybridized with a corresponding preparation froma cancer cell provides a means of directly determining cancer-relateddifferences in copy number of chromosomal regions.

“Targeted therapeutics” is used herein to denote any therapeuticmodality that affects only or primarily only the cells or tissuesselected (“targeted”) for treatment. A monoclonal antibody specific foran antigen expressed only by a target (if retained by the target) ishighly useful in targeted therapeutics. In the case of unwanted cellssuch as cancer cells, if the antibody doesn't induce destruction of thetarget directly, it may do so indirectly by carrying to the target, forexample, an agent coupled to the antibody. On the other hand, agentsthat suppress processes that tend to promote uncontrolled proliferationof cells (“antineoplastic agents”) can be delivered to target sites inthis manner.

The term “agent” is used herein in its broadest sense to refer to acomposition of matter, a process or procedure, a device or apparatusemployed to exert a particular effect. By way of non-limiting example, asurgical instrument may be employed by a practitioner as an “excising”agent to remove tissue from a subject; a chemical may be used as apharmaceutical agent to remove, damage or neutralize the function of atissue, etc. Such pharmaceutical agents are said to be “anticellular.”Cells may be removed by an agent that promotes apoptosis. A variety oftoxic agents, including other cells (e.g., cytotoxic T-cell lymphocytes)and their secretions, and a plethora of chemical species, can damagecells.

The term “by-stander”, as used herein, refers to a process or eventinitiated or affected by another, causative event or process

The term “knockdown”, as used herein, refers to a method of selectivelypreventing the expression of a gene in an individual.

The term “oncogene”, as used herein, refers to any gene that regulates aprocess affecting the suppression of abnormal proliferative events.

The term “single nucleotide polymorphism” or “SNP”, as used herein,refers to a DNA sequence variation occurring when a single nucleotide inthe genome (or other shared sequence) differs between members of aspecies or between paired chromosomes in an individual. Singlenucleotide polymorphisms may fall within coding sequences of genes,non-coding regions of genes, or in the intergenic regions between genes.Single nucleotide polymorphisms within a coding sequence will notnecessarily change the amino acid sequence of the protein that isproduced, due to degeneracy of the genetic code. A Single nucleotidepolymorphism in which both forms lead to the same polypeptide sequenceis termed synonymous (sometimes called a silent mutation)—if a differentpolypeptide sequence is produced they are non-synonymous. Singlenucleotide polymorphisms that are not in protein-coding regions maystill have consequences for gene splicing, transcription factor binding,or the sequence of non-coding RNA.

The term “tissue array” or “tissue microarray”, as used herein, refersto high throughput platforms for the rapid analysis of protein, RNA, orDNA molecules. These arrays can be used to validate the clinicalrelevance of potential biological targets in the development ofdiagnostics, therapeutics and to study new disease markers and genes.Tissue arrays are suitable for genomics-based diagnostic and drug targetdiscovery.

As used herein, the term “shRNA” or “short hairpin RNA” refers to asequence of ribonucleotides comprising a single-stranded RNA polymerthat makes a tight hairpin turn on itself to provide a “double-stranded”or duplexed region. shRNA can be used to silence gene expression via RNAinterference. shRNA hairpin is cleaved into short interfering RNAs(siRNA) by the cellular machinery and then bound to the RNA-inducedsilencing complex (RISC). It is believed that the complex inhibits RNAas a consequence of the complexed siRNA hybridizing to and cleaving RNAsthat match the siRNA that is bound thereto.

As used herein, the term “RNA interference” or “RNAi” refers to thesilencing or decreasing of gene expression by siRNAs. It is the processof sequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi inhibits the gene by compromising the function of a target RNA,completely or partially. Both plants and animals mediate RNAi by theRNA-induced silencing complex (RISC); a sequence-specific,multicomponent nuclease that destroys messenger RNAs homologous to thesilencing trigger. RISC is known to contain short RNAs (approximately 22nucleotides) derived from the double-stranded RNA trigger, although theprotein components of this activity are unknown. However, the22-nucleotide RNA sequences are homologous to the target gene that isbeing suppressed. Thus, the 22-nucleotide sequences appear to serve asguide sequences to instruct a multicomponent nuclease, RISC, to destroythe specific mRNAs. Carthew has reported (Curr. Opin. Cell Biol. 13(2):244-248 (2001)) that eukaryotes silence gene expression in the presenceof dsRNA homologous to the silenced gene. Biochemical reactions thatrecapitulate this phenomenon generate RNA fragments of 21 to 23nucleotides from the double-stranded RNA. These stably associate with anRNA endonuclease, and probably serve as a discriminator to select mRNAs.Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart.

As used herein, the term “siRNAs” refers to short interfering RNAs. Insome embodiments, siRNAs comprise a duplex, or double-stranded region,of about 18-25 nucleotides long; often siRNAs contain from about two tofour unpaired nucleotides at the 3′ end of each strand. At least onestrand of the duplex or double-stranded region of a siRNA issubstantially homologous to or substantially complementary to a targetRNA molecule. The strand complementary to a target RNA molecule is the“antisense strand”; the strand homologous to the target RNA molecule isthe “sense strand”, and is also complementary to the siRNA antisensestrand. siRNAs may also contain additional sequences; non-limitingexamples of such sequences include linking sequences, or loops, as wellas stem and other folded structures. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants.

The term “xenograft”, as used herein, refers to the transfer ortransplant of a cell(s) or tissue from one species to an unlike species(or genus or family).

The term “orthotopic” or “orthotopic xenograft”, as used herein, refersto a cell or tissue transplant grafted into its normal place in thebody.

The term “fluorescent activated cell sorting” or “FACS”, as used herein,refers to a technique for counting, examining, and sorting microscopicparticles suspended in a stream of fluid. It allows simultaneousmultiparametric analysis of the physical and/or chemical characteristicsof single cells flowing through an optical and/or electronic detectionapparatus. Generally, a beam of light (usually laser light) of a singlewavelength is directed onto a hydro dynamically focused stream of fluid.A number of detectors are aimed at the point where the stream passesthrough the light beam; one in line with the light beam (ForwardScatter, correlates to cell volume) and several perpendicular to thebeam, (Side Scatter, correlates to the inner complexity of the particleand/or surface roughness) and one or more fluorescent detectors. Eachsuspended particle passing through the beam scatters the light in someway, and fluorescent chemicals found in the particle or attached to theparticle may be excited into emitting light at a lower frequency thanthe light source. By analyzing the combinations of scattered andfluorescent light picked up by the detectors it is then possible toderive information about the physical and chemical structure of eachindividual particle.

The term “data mining”, as used herein, refers to the automated orconvenient extraction of patterns representing knowledge implicitlystored or captured in large databases, data warehouses, internetwebsites, other massive information repositories, or data streams.

The terms “over-express”, “over-expressing” and grammatical equivalents,as used herein, refer to the production of a gene product at levels thatexceed production in normal or control cells. The term “over-expression”or “highly expressed” may be specifically used in reference to levels ofmRNA to indicate a higher level of expression than that typicallyobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA are measured using any of a number of techniques known to thoseskilled in the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed, theamount of 28S rRNA (an abundant RNA transcript present at essentiallythe same amount in all tissues) present in each sample can be used as ameans of normalizing or standardizing the mRNA-specific signal observedon Northern blots. Over-expression may likewise result in elevatedlevels of proteins encoded by said mRNAs.

The term “heatmap”, as used herein, refers to a graphical representationof data where the values obtained from a variable two-dimensional mapare represented as colors. As related to the field of molecular biology,heat maps typically represent the level of expression of multiple genesacross a number of comparable samples as obtained from a microarray.

The term “phage display”, as used herein, refers to theintegration/ligation of numerous genetic sequences from a DNA library,consisting of all coding sequences of a cell, tissue or organism libraryinto the genome of a bacteriophage (i.e. phage) for high-throughputscreening protein-protein and/or protein-DNA interactions. Using amultiple cloning site, these fragments are inserted in all threepossible reading frames to ensure that the cDNA is translated. DNAfragments are then expressed on the surface of the phage particle aspart of it coat protein. The phage gene and insert DNA hybrid is thenamplified by transforming bacterial cells (such as TG1 E. coli cells),to produce progeny phages that display the relevant protein fragment aspart of their outer coat. By immobilizing relevant DNA or proteintarget(s) to the surface of a well, a phage that displays a protein thatbinds to one of those targets on its surface will remain while othersare removed by washing. Those that remain can be eluted, used to producemore phage (by bacterial infection with helper phage) and so produce anenriched phage mixture. Phage eluted in the final step can be used toinfect a suitable bacterial host, from which the phagemids can becollected and the relevant DNA sequence excised and sequenced toidentify the relevant, interacting proteins or protein fragments.

The term “apoptosis”, as used herein, refers to a form of programmedcell death in multicellular organisms that involves a series ofbiochemical events that lead to a variety of morphological changes,including blebbing, changes to the cell membrane such as loss ofmembrane asymmetry and attachment, cell shrinkage, nuclearfragmentation, chromatin condensation, and chromosomal DNAfragmentation. Defective apoptotic processes have been implicated in anextensive variety of diseases; for example, defects in the apoptoticpathway have been implicated in diseases associated with uncontrolledcell proliferations, such as cancer.

The term “bioluminescence imaging” or “BLI”, as used herein, refers tothe noninvasive study of ongoing biological processes in livingorganisms (for example laboratory animals) using bioluminescence, theprocess of light emission in living organisms. Bioluminescence imagingutilizes native light emission from one of several organisms whichbioluminescence. The three main sources are the North American firefly,the sea pansy (and related marine organisms), and bacteria likePhotorhabdus luminescens and Vibrio fischeri. The DNA encoding theluminescent protein is incorporated into the laboratory animal eithervia a virus or by creating a transgenic animal. While the total amountof light emitted via bioluminescence is typically small and not detectedby the human eye, an ultra-sensitive CCD camera can imagebioluminescence from an external vantage point. Common applications ofBLI include in vivo studies of infection (with bioluminescentpathogens), cancer progression (using a bioluminescent cancer cellline), and reconstitution kinetics (using bioluminescent stem cells).

The term “consensus region” or “consensus sequence”, as used herein,refers to the conserved sequence motifs that show which nucleotideresidues are conserved and which nucleotide residues are variable whencomparing multiple DNA, RNA, or amino acid sequence alignments. Whencomparing the results of a multiple sequence alignment, where relatedsequences are compared to each other, and similar functional sequencemotifs are found. The consensus sequence shows which residues areconserved (are always the same), and which residues are variable. Aconsensus sequence may be a short sequence of nucleotides, which isfound several times in the genome and is thought to play the same rolein its different locations. For example, many transcription factorsrecognize particular consensus sequences in the promoters of the genesthey regulate. In the same way restriction enzymes usually havepalindromic consensus sequences, usually corresponding to the site wherethey cut the DNA. Splice sites (sequences immediately surrounding theexon-intron boundaries) can also be considered as consensus sequences.In one aspect, a consensus sequence defines a putative DNA recognitionsite, obtained for example, by aligning all known examples of a certainrecognition site and defined as the idealized sequence that representsthe predominant base at each position. Related sites should not differfrom the consensus sequence by more than a few substitutions.

The term “linkage”, or “genetic linkage,” as used herein, refers to thephenomenon that particular genetic loci of genes are inherited jointly.The “linkage strength” refers to the probability of two genetic locibeing inherited jointly. As the distance between genetic loci increases,the loci are more likely to be separated during inheritance, and thuslinkage strength is weaker.

The term “neighborhood score”, as used herein, refers to the relativevalue assigned to a genomic locus based on a geometry-weighted sum ofexpression scores of all the genes on a given chromosome, as ameasurement of the copy number status of the locus. A positiveneighborhood score is indicative of an increase in copy number, whereasa negative neighborhood score is indicative of a decrease in copynumber.

The term “expression score”, as used herein, refers to the expressiondifferences (i.e., the level of transcription (RNA) or translation(protein)) between comparison groups on a given chromosome. Theexpression score for a given gene is calculated by correlating the levelof expression of said gene with a phenotype in comparison. For example,an expression score may represent a comparison of the expressiondifferences of a given gene in normal vs. abnormal conditions, such asparental vs. drug-resistant cell lines. As used herein, the term“regional expression score” refers to the expression score of gene(s) inproximity to the locus in consideration. Since linkage strength betweengenetic loci decreases (i.e. decays) as the distance between themincreases, the “regional expression score” more accurately reflects theexpression differences between comparison groups by assigning greaterweight to the expression scores of genes in proximity to the locus inconsideration.

The terms “geometry-weighted” or “geometry-weighted sum”, as usedherein, refers to the significance attached to a given value, forexample an “expression score”, based on physical position, including butnot limited to genomic position. Since linkage strength between geneticloci decreases (i.e. decays) as the distance between them increases, the“weight” assigned to a given value is adjusted accordingly.

The term “copy number alteration” or “CNA”, as used herein, refers tothe increase (i.e. genomic gain) or decrease (i.e. genomic loss) in thenumber of copies of a gene at a specific locus of a chromosome ascompared to the “normal” or “standard” number of copies of said genethat locus. As used herein, an increase in the number of copies of agiven gene at a specific locus may also be referred to as an“amplification” or “genomic amplification” and should not be confusedwith the use of the term “amplification” as it relates, for example, toamplification of DNA or RNA in PCR and other experimental techniques.

The term “clonogenic assay”, as used herein, refers to a technique forstudying whether a given cancer therapy (for example drugs or radiation)can reduce the clonogenic survival and proliferation of tumor cells.While any type of cell may be used, human tumor cells are commonly usedfor ontological research. The term “clonogenic” refers to the fact thatthese cells are clones of one another.

The term “adjuvant therapy”, as used herein, refers to additionaltreatment given after the primary treatment to increase the chances of acure. In some instances, adjuvant therapy is administered after surgerywhere all detectable disease has been removed, but where there remains astatistical risk of relapse due to occult disease. If known disease isleft behind following surgery, then further treatment is not technically“adjuvant”. Adjuvant therapy may include chemotherapy, radiationtherapy, hormone therapy, or biological therapy. For example,radiotherapy or chemotherapy is commonly given as adjuvant treatmentafter surgery for a breast cancer. Oncologists use statistical evidenceto assess the risk of disease relapse before deciding on the specificadjuvant therapy. The aim of adjuvant treatment is to improvedisease-specific and overall survival. Because the treatment isessentially for a risk, rather than for provable disease, it is acceptedthat a proportion of patients who receive adjuvant therapy will alreadyhave been cured by their primary surgery. Adjuvant chemotherapy andradiotherapy are often given following surgery for many types of cancer,including colon cancer, lung cancer, pancreatic cancer, breast cancer,prostate cancer, and some gynecological cancers.

The term “matched samples”, as used herein, as for example “matchedcancer samples” refers to a sample in which individual members of thesample are matched with every other sample by reference to a particularvariable or quality other than the variable or quality immediately underinvestigation. Comparison of dissimilar groups based on specifiedcharacteristics is intended to reduce bias and the possible effects ofother variables. Matching may be on an individual (matched pairs) or agroup-wide basis.

The term “genomic segments”, as used herein, refers to any defined partor region of a chromosome, and may contain zero, one or more genes.

The teen “co-administer”, as used herein, refers to the administrationof two or more agents, drugs, and/or compounds together (i.e. at thesame time).

The term “diagnose” or “diagnosis”, as used herein, refers to thedetermination, recognition, or identification of the nature, cause, ormanifestation of a condition based on signs, symptoms, and/or laboratoryfindings.

The term “resistance”, as used herein, refers to cancer cells that donot respond to chemotherapy drugs (i.e. chemotherapeutic agents).Typically, a first course of chemotherapy may prove highly beneficial,nearly annihilating a tumor, but a few resistant cancer cells oftensurvive and proliferate. Too often, despite more aggressive second andthird courses of chemotherapy, the remaining drug-defiant cells thrive,displaying increasing resistance to drug therapy and eventuallydisplaying virtual invulnerability to chemotherapy. After the drug'seffectiveness fades, the patient relapses. This occurs in patients witha variety of blood cancers and solid tumors, including breast, ovarian,lung, and lower gastrointestinal tract cancers. Nature Biotechnology18:IT18-IT20 (2000). Resistance to treatment with anticancer drugsresults from a variety of factors including individual variations inpatients and somatic cell genetic differences in tumors, even those fromthe same tissue of origin. Frequently resistance is intrinsic to thecancer, but as therapy becomes more and more effective, acquiredresistance has also become common. The development of multidrugresistance (MDR) to chemotherapy remains a major challenge in thetreatment of cancer. Resistance exists against every effectiveanticancer drug and can develop by numerous mechanisms includingdecreased drug uptake, increased drug efflux, activation of detoxifyingsystems, activation of DNA repair mechanisms, and insensitivity todrug-induced apoptosis. Methods Mol. Biol. 596:47-76 (2010).

In some embodiments, the present invention contemplates treating drugresistant cancer cells. It is not intended that the present invention belimited to the degree of resistance, i.e. resistance can be shown simplyby the fact that it takes higher doses of drug to kill these cells. Thecells need not be resistant at every dose. The cells may be resistantsuch that higher doses needed to kill the cells will not be welltolerated by the patient.

As used herein, “Doxorubicin” (trade name Doxil) also known as“hydroxydaunorubicin” or “Adriamycin” refers to a drug used in cancerchemotherapy, that is considered to be the most effective agent in thetreatment of breast cancer patients. Doxorubicin is an anthracyclineantibiotic, closely related to the natural product daunomycin, and likeall anthracyclines, works by intercalating DNA, with the most seriousadverse effect being life-threatening heart damage. Doxorubicin iscommonly used in the treatment of a wide range of cancers, includingsome leukemia's and Hodgkin's lymphoma, as well as cancers of thebladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma,multiple myeloma. It is frequently used in breast cancer therapy eitheras single-agent or in combination with other drugs like docetaxel andcyclophosphamide. Unfortunately, resistance to this agent is common,representing a major obstacle to successful treatment. Mol. Cancer.Ther. 5(8):2115-20 (2006). Doxorubicin is administered intravenously, asthe hydrochloride salt. It may be sold under the brand names AdriamycinPFS, Adriamycin RDF, or Rubex. Commonly used doxorubicin-containingregimens include, but are not necessarily limited to, AC (Adriamycin,cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin,vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin,cyclophosphamide, vincristine, procarbazine, prednisone), BEP(bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF(cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV(cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide,Adriamycin, vincristine, prednisone), ChlVPP/EVA (chlorambucil,vincristine, procarbazine, prednisone, etoposide, vinblastine,Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine, Adriamycin,dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin orplatinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil,Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin,adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone),MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin,cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin),ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide,etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin,cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine,methotrexate, leucovorin), VAD (vincristine, Adriamycin, dexamethasone),Regimen I (vincristine, Adriamycin, etoposide, cyclophosphamide) andVAPEC-B (vincristine, Adriamycin, prednisone, etoposide,cyclophosphamide, bleomycin).

Analogues of Doxorubicin for cancer chemotherapy include, but are notlimited to, daunorubicin, 4-demethoxydaunorubicin (idarubicin),pirarubicin (DaunoXome), epirubicin, pegylated liposomal doxorubicin(Lipo-Dox®), antibody-conjugated liposomal doxorubicin (e.g.S5A8-Lipo-Dox), 4′-epidoxorubicin, AD198,N-(5,5-Diacetoxypent-1-yl)doxorubicin, and Doxorubicin analogues 2-5,incorporating the following alkylating or latent alkylatingsubstituents, R, on the 3′-position of the daunosamine sugar. 2,R═NHCOC₆H₄(p)SO₂F; 3, R═NHCOCH₂Br; 4, R═NHCOCH₂C1; 5,R═NHCON(NO)CH₂CH₂Cl. J Med. Chem. 1991 Feb.; 34(2):561-4.

As used herein, “Ibrutinib”, also known as PCI-32765, refers to a drugfor the treatment of various types of hematopoietic related cancer.However, in one embodiment, the present invention contemplates the useof ibrutinib for non-hematopoietic related cancers, and in particularfor breast cancer.

DETAILED DESCRIPTION

Tyrosine kinases (TKs) catalyze the reversible process of tyrosinephosphorylation, a key step in most signal transduction pathways thatgovern cellular proliferation, survival, differentiation, and motility.Dysregulation of TKs, as occurs through inappropriate expression,activation, or both, is commonly associated with human cancers(Blume-Jensen and Hunter 2001; Giamas, et al. 2010). As a result, TKs,as a class, are the most commonly found dominant oncogenes (Baselga2006; Blume-Jensen and Hunter 2001; Krause and Van Etten 2005; Vassilevand Uckun 2004). Receptor protein tyrosine kinases (RPTKs) transmitextracellular signals across the plasma membrane to cytosolic proteins,stimulating the formation of complexes that regulate key cellularfunctions. Over half of the 90 tyrosine kinases have been implicated inhuman cancers and are for this reason considered highly promising drugtargets. To gain insight into the tyrosine kinases that contribute tobreast cancer related cellular mechanisms, we carried out a large-scaleloss-of-function analysis of the tyrosine kinases, using RNAinterference, in the clinically relevant Erb-B2 positive, BT474 breastcancer cell line. The cytosolic, non-receptor tyrosine kinase Bruton'styrosine kinase (BTK), which has been extensively studied for its rolein B cell development, was among those tyrosine kinase genes requiredfor BT474 breast cancer cell survival. The BTK protein identified was analternative faun containing an amino-terminal extension. Thisalternative form of the Btk message is also present in tumorigenicbreast cells at significantly higher levels than in normal breast cells.

Small molecules that directly inhibit the catalytic activity of tyrosinekinases have been sought as potential cancer chemotherapeutics. Recentsuccesses with a few well-studied tyrosine kinases have proven the valueof these proteins as drug targets. Imatinib mesylate (Gleevec) hasproven hugely successful in treating CML. The EGFR inhibitors, Gefitinib(Iressa) and erlotinib (Tarceva), are currently used on a variety ofsolid tumors (Krause and Van Etten 2005; Kris, et al. 2003; Shepard, etal. 2008). Trastuzumab (Herceptin), a humanized monoclonal antibody thatspecifically inhibits Erb-B2, is widely used in the treatment of breastcancers. Each of these treatments, however, has significant limitationsrelated to tissue spectrum, acquired resistance, and efficacy inadvanced disease (Nahta and Esteva 2006). The identification ofadditional TK genes and pathways that contribute to the survival ofdistinct cancer cell types, so that they can be effectively targeted,would be of great value.

A large-scale RNAi screen found that nearly ⅓ (30%) of the human TKsscreened impeded cellular proliferation by more than half of controllevels in an ErbB-2 over-expressing breast cancer cell line. Amongthese, 54% were receptor TKs and 46% were non-receptor cytoplasmic TKsand with few exceptions, were distinct from those identified as survivalkinases in an RNAi screen carried out in HeLa cells (MacKeigan, et al.2005). This may reflect decreased cellular proliferation or increasedcell death in BT474 breast cancer cells. Unexpectedly, four of the fivenon-receptor tyrosine kinases that exhibited the strongest impact oncellular proliferation were members of the Tec family of cytoplasmictyrosine kinases. Surprisingly, a novel isoform of a member of the Tecfamily of non-receptor tyrosine kinases, Bruton's tyrosine kinase (BTK),which is known primarily for its critical role in B cell maturation, isamong the TKs that exhibited the strongest impact on cellularproliferation. The expression of this novel BTK isoform is elevated in anumber of breast cancer cell lines compared to non-tumorigenic breastcell lines. Further exploration of one Tec family member, Bruton'sTyrosine Kinase, (BTK) revealed that its knockdown using either siRNAsled to an increase in apoptosis. A unique Btk transcript was isolatedfrom BT474 cells, which encodes an additional 34 amino acids in framewith the published BTK start codon, suggesting that an N-terminallyelongated form of the BTK protein is present in BT474 breast cancercells. The expression of this novel Btk transcript is higher in a numberof breast cancer cell lines compared to non-tumorigenic breast celllines. These results suggest that an alternative BTK protein,potentially with other Tec family tyrosine kinases, contribute to breastcancer cell survival.

The validity of the breast screen is supported by the identification ofseveral kinases with known transformative properties. ErbB-2, inparticular was expected to be required for BT474 proliferation since itis amplified and constitutively activated in this breast cancer cellline. Other TK's identified in the screen have established roles inbreast cancer. FGFR2 is amplified or over-expressed in 5-10% of breasttumors (Adnane, et al. 1991; Cha, et al. 2008; Penault-Llorca, et al.1995), and has been the focus of several genome-wide association studiescovering thousands of unique breast tumors (Hunter, et al. 2007).NTRK2/TRKB is the brain-derived neurotropic factor (BDNF) receptor andis expressed in a subset of high-grade human breast tumors (Cameron andFoster 2008).

The major and largely unexpected discovery of the screen was theidentification of a functional role for the cytosolic, non-receptortyrosine kinase BTK in breast cancer cell survival. BTK is thought tofunction primarily in cells derived from the hematopoietic cell lineage,where it is crucial for B cell maturation. Although a recent study hasindicated that the expression of this kinase is elevated in a subset ofbasal breast tumors due to the presence of tumor-infiltrativelymphocytes (Sabatier, et al. 2011), our study focused on tumor cellsInhibition of BTK using RNAi or pharmacological inhibitors results inincreased apoptosis in BT474 and MCF-7 breast cancer cells (FIGS. 10A,B; FIG. 13B). Importantly, RNAi-triggers specific to the alternateisoform, BTK-C, which is expressed in breast cancer cells compromisecell survival; BTK-C over-expression inhibits apoptosis induced byDoxorubicin (FIG. 13D, E), both indicating that at least some of thepro-survival effects of BTK are due to this isoform.

Although no role has previously been described for BTK in epithelialcell cancers, dysregulated BTK expression is associated with theformation and maintenance of leukemias and lymphomas and is currentlybeing targeted therapeutically. BTK over-expression has been implicatedin imatinib resistance to chronic myeloid leukemia (CML) and acutelymphoblastic leukemia (ALL) (Villuendas, et al. 2006); (Hofmann, et al.2002). Its constitutive activation due to deregulated B cell receptor(BCR) engagement contributes to the genesis of B cell lymphomas (Irish,et al. 2006); (Kuppers 2005) and new inhibitors are currently inclinical trials for lymphoid malignancies (Honigberg, et al. 2010;Winer, et al. 2012). It is worth noting that a number of tyrosine kinasegenes related to B cell signaling were also identified in the screen asbeing important for the proliferation or survival of BT474 cells. Theseinclude LYN which induces BTK phosphorylation and ABL2 (Lin, et al.2009) which may regulate BTK (Backesjo, et al. 2002) among others.

The BTK protein expressed in breast cells has an amino-terminal 34 aminoacid extension, which we have termed BTK-C. Higher levels of this BTK-CmRNA are found in breast cancer cells compared to non-cancerous breastcells. Early experimentation has not detected functional differencesbetween the BTK-A and BTK-C proteins. It may be that deregulation of analternative promoter in breast cancer cells causes increased expressionof BTK-C and that this provides an essential function for these cells.In this sense, BTK-C is similar to other cancer related genes. Recentwork has shown that alternative promoter usage in genes involved incancer initiation and progression are significantly more likely to havemultiple promoters than are non-cancer causing genes (Davuluri, et al.2008). For example, the aberrant use of an alternative promoter in theTGFf33, LEF1, and CyP19A1 genes have been directly linked to cancer cellgrowth (Archey, et al. 1999; Harada, et al. 1993; Li, et al. 2006). Morerecently, the expansion of genomic platforms has led to genome-wideviews of promoter usage that have revealed increased alternativepromoter usage in tumor cells compared to normal cells (Thorsen, et al.2011).

At present, the mechanism for BTK supporting breast cancer cell survivalis unknown. BTK, like several kinases identified in the screen, hasmultiple protein-protein/protein-lipid interaction domains, enabling theformation of numerous and diverse signal complexes. This complexity hasmade its mechanism of action in B cells where it is relatively wellstudied poorly understood. In breast cells, microarray analysisindicates that BTK may affect transcription of specific targets. Amongtranscripts more than 2.5 fold upregulated by BTK-C compared to BTK-Awere the calcium handling proteins calbindin (CALB1) and troponin(TNNI2) which may suggest a role for this kinase in calcium signaling asoccurs in B cells, and STEAP4 which implicates BTK-C in the increasedglucose uptake found in a number of breast cancer cells (FIG. 15). Inmany ways, these findings are similar to recent studies that indicatethat other B cell kinases, including Abl (Srinivasan and Plattner 2006)(Srinivasan, et al. 2008), BMX (Dai, et al. 2006; Jiang, et al. 2007)and Syk (Ruschel and Ullrich 2004), have critical functions in solidtumors and not just haematological malignancies. In these cases themechanisms are incompletely understood and may involve pathwaysdifferent from those operating in B cells. Further experimentation isnecessary for the elucidation of how BTK and the other B cell kinasesbecome integrated in epithelial cell signal transduction pathways.

RNAi knockdown screen of the PTKS in BT474 breast cancer cells. Afunctional genomic approach was taken to evaluate the contribution ofeach TK to breast cancer cell viability. An unbiased functional RNAiscreen targeting the PTKs in the clinically relevant ErbB-2 positive,BT474 breast cancer cell line was performed to identify additional TKsthat when knocked down, sensitized the cells to cell death. 236short-hairpin RNAs (shRNAs) (Paddison et al., 2004; Silva et al. 2005)were used to target 82 of the 90 PTK genes in the ErbB-2-positive breastcancer cell line BT474, such that, on average, each PTK was targetedwith 3 independent shRNA constructs. Each shRNA was co-transfected witha plasmid that directs the expression of GFP so that differences intransfection could be normalized. Effects on cells were monitored usingalamarBlue (Biosource), a fluorimetric indicator of both cellproliferation and viability that has proven useful in RNAi screeens(Kourtidis et al., 2007), (FIG. 1). 25 of the 82 genes (30%) whensilenced by shRNAs led to a fifty percent or greater decrease in BT474cellular proliferation compared to control levels, in three replicateexperiments using at least two unique shRNAs per gene. EGFR, ERBB2,ABL2, FES, NTRK2 (TRK-B), PTK2B, FGFR2, LYN (V-yes-1), EphA1, and BTKwere among the kinases that when knocked down caused the greatestreduction in BT474 cellular proliferation levels (Table 2). The validityof the screen is supported in that many of these PTKs have previouslydescribed roles in breast tumors (EGFR, ERBB2, FGFR2, PTK2B,NTRK2/TRK-B, EphA1, ABL2) (Behmoaram, et al. 2008; Brantley-Sieders, etal. 2005; Chan, et al. 2006; Ogawa, et al. 2000; Srinivasan and Plattner2006). These results support the validity of the screen in identifyingkinases that are important for breast cancer cell proliferation orsurvival. For instance, over-expression of ERBB2 in mammary epithelialcells causes malignant transformation and amplification of ERBB2 ininvasive primary breast cancers correlates with reduced patient survival(Baselga_(—)2006_Science). FGFR2 is amplified or over-expressed in 5-10%of breast tumors (Adnane et al., 1991; Cha et al., 2008; Penault-Llorcaet al., 1995), and has been the focus of several genome-wide associationstudies covering thousands of unique breast tumors (Hunter et al.,2007). NTRK2/TRKB is the brain-derived neurotrophic factor (BDNF)receptor that is expressed in a subset of high-grade human breast tumors(Cameron and Foster, 2008).

Btk Silencing Leads to Increased Apoptosis. Bruton's Tyrosine Kinase(BTK) was among those genes whose knockdown caused the most significantreduction in BT474 cellular proliferation (FIG. 1; Table 2). This issurprising since BTK is thought to function primarily in cells derivedfrom the hematopoietic cell lineage (de Weers, et al. 1993; Smith, etal. 1994). Mutations in the human Btk gene cause inherited X-linkedagammaglobulinemia which is characterized by a virtual absence of Blymphocytes. This is due to a block between the pro- and pre-B cellstages of B cell maturation (Tsukada et al., 1993); (Lindvall et al.,2005). Btk over-expression has been implicated in imatinib resistance tochronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL)(Villuendas et al., 2006); (Hofmann et al., 2002). Its constitutiveactivation due to deregulated B cell receptor (BCR) engagement is anintegral component to certain B cell lymphomas (Irish et al., 2006);(Kuppers, 2005). In B-lineage lymphoid cells, Btk serves a protectiverole through inhibition of Fas/APO-1 mediated apoptosis (Qiu and Kung,2000; Vassilev et al., 1999).

We chose to interrogate the role Btk might have in breast cancer cellsfurther since no function had been previously described for it in eithernormal or malignant breast cellular processes. Quantitative PCR (qPCR)analysis detected the Btk transcript towards the later rounds of cycling(34th of 40 total cycles) indicating that Btk levels in BT474 cells arerelatively low. Nevertheless, the Btk transcript could be specificallyknocked down 2.2 fold further using siRNAs (data not shown). The initialshRNA screen, utilizing a redox indicator assay, revealed a severedecrease in cellular proliferation after knockdown of Btk in BT474cells. To determine if reduced proliferation correlated with an increasein apoptosis BT474 cells were transfected with siRNAs targeting Btk andcleaved caspase 3 levels were compared to control cells. BT474 cellsthat were transfected with the Btk siRNA had an 11 fold increase inapoptotic cells compared to control (FIG. 2A-2C) indicating that theloss of proliferation in Btk silenced BT474 cells is due, at least inpart, to increased apoptosis.

The shRNA screen and additional siRNA silencing experiments reveal asignificant decrease in cellular proliferation after knockdown of BTK inBT474 cells (FIG. 1, FIG. 10). Knockdown of BTK in BT474 cells withsiGENOME SMART pool duplex siRNAs, which are transfected into thesecells more efficiently, leads to widespread cell death. In these cells,BTK transcript levels are specifically knocked down to 37.5% of controlat 48 hrs (not shown) prior to the significant loss of cells that occursbetween 72 and 96 hours and that is due to increased apoptosis. BT474cells transfected with siRNAs targeting BTK have an 11-fold increase inapoptotic cells compared to control as evidenced by increased cleavedcaspase 3 levels (FIG. 10A, B). Similarly, knockdown of BTK has the sameeffect increasing levels of apoptosis in MCF-7 cells. This indicatesthat the decrease in proliferative activity found in BTK-silenced BT474cells is due, at least in part, to increased apoptosis. Since BTKknockdown with both shRNAs and several siRNAs all result in a similarapoptotic effect, we are confident this result is due to silencing ofBTK and is not an off-target effect.

A novel form of the btk message is present in breast cancer cells.Although no function has been previously described for Btk in breastcells, inhibiting Btk using shRNAs severely reduced the proliferation ofthe BT474 breast cancer cell line compared to control (FIG. 1). Thepresence of the Btk transcript in BT474 breast cancer cells wasconfirmed by amplifying the transcript from cDNA. Two distinct Btkspecific primer sets were generated by designing primer pairs to uniqueregions of the Btk gene at nucleotide positions 142 and 2,522, (Btk5′UTR) and 519 and 2,079, (Btk internal) (Table 1). The Btk internalprimer set is specific to sites located between the translational startand stop codons while the Btk 5′UTR forward primer is located 22 bpsupstream from the translational start codon, within the 5′UTR, and thedownstream primer is located 379 bps downstream from the translationalstop codon. Interestingly, while a product of the expected size wasamplified from BT474 cDNA using the Btk internal forward primer witheither of the reverse primers (FIG. 8 and data not shown), no productwas amplified from BT474 cDNA when the Btk 5′UTR forward primer was usedwith either of the reverse primers (FIG. 8). The 5′UTR primer set didgenerate a product of the expected size from a positive control cDNAsample generated from Namalwa B-cells (FIG. 8), suggesting thatamino-terminal sequence of the Btk transcript in BT474 cells differedfrom the published sequence.

BT474 breast cancer cells express an alternative form of the btk messagefrom an alternative promoter. To confirm the full length Btk product inBT474 cells, sequence information was obtained upstream from thepublished Btk start codon using rapid amplification of cDNA ends(5′RACE). A sequence alignment that included the first 395 nucleotidesof the published Btk exon 1 sequence (accession # U3399) and the 398nucleotides obtained using 5′ RACE revealed that while the sequenceswere 100% identical from position 307 downstream, the sequence upstreamof position 307 was non-homologous (FIG. 3A).

The Btk sequence obtained from BT474 cells using 5′RACE is 100%identical to two entries in the genome database (Levy et al., 2007)(Griffiths-Jones, 2004) that were derived using an automated analysisfor gene prediction program (GNOMON). The sequence is named Btk-cra-C(hereafter referred to as Btk-C) reflecting its status as an automatedcomputational prediction rather than an experimentally verified genemessage. In addition, the Btk-C sequence is 100% identical to twosequences in the expressed sequence tag (EST) database. The first of thetwo EST sequences was obtained from a human pheochromocytoma tissuesample (Yang, Y. et al. 2000, unpublished; accession # AV733045) and thesecond from a study seeking to identify putative alternative promotersof human genes from human peripheral blood mononuclear cells (PEBLM2),(Kimura et al., 2006).

The unique, first exon present in the Btk-C message is located 4,416 bpsto the 3′ side of the first exon from the published Btk gene (Btk-cra-A,hereafter referred to as Btk-A) and 255 bps to the 5′ side of theribosomal protein L36a (FIG. 3B, C). Evidence of additional ESTs thatare identical to the full-length Btk-C transcript, along with theidentification of putative pol II promoter sites (Ref) and transcriptionfactor binding sites including Ets, Ap2, AhR HoxA7 (Matys et al., 2003),strongly suggests that the Btk-C transcript found in BT474 cells isdriven by an alternate promoter located 4,416 bps to the 3′ side if thepublished Btk-A promoter. In addition, the first exons of the twotranscripts must use different donor sites to yield the mature RNAs(FIG. 3A-3C). Furthermore, translation of the Btk-C nucleotide sequenceinto amino acids revealed an additional 47 amino acids that when alignedto the BTK-A amino acid sequence is in frame with the BTK-A methioninestart codon. Importantly, the additional 47 amino acid stretch presentin the Btk-C mRNA from BT474 breast cancer cells contains two additionalmethionine codons, at nucleotide positions 241-243 and 265-267,respectively (FIG. 3A), creating a putative elongated BTK protein (FIG.4A). Bioinformatic analyses (ExPASy Proteomics Tools) of the BTK-Cadditional amino acid sequence for conserved motifs including, cleavagesites, phosphorylation, N-glycosylation, or Nmyristoylation sites didnot identify any putative functional roles. Although, neither of thenovel translational start sites contains strongly conserved Kozaksequences, a transcription start site prediction program (Down andHubbard, 2002) identified a putative transcription start site, locatedwithin a CpG island, 200 bps upstream from the start of the Btk-C mRNA.Additionally, promoter prediction analyses (Knudsen, 1999) of 2500nucleotides of the Btk-C sequence, located just upstream from thetranscription start site (TSS), has predicted the presence of twoputative promoters. The first is located 823 bps upstream from the Btk-CTSS and is predicted to be a highly likely promoter with a score of1.156 and the second is located 22 nucleotides upstream from the Btk-CTSS and is predicted to be a promoter with marginal likeliness with ascore of 0.699. A similar promoter prediction analyses using the 2500nucleotides located just upstream from the Btk-A TSS has also predictedthe presence of two putative promoters, but at greater distances fromthe TS S and with lower likeliness scores compared to either of theBtk-C predicted promoters. The first predicted Btk-A promoter is located867 bps upstream from the Btk-A TSS and has a marginal likeliness scoreof 0.649 and the second is located 388 bps upstream from the Btk-A TSSwith a marginal likeliness score of 0.569.

Consistent with the bioinformatic TSS and promoter prediction analysesof the Btk-C message, western blotting of BT474 total lysate using a BTKspecific antibody detected a faint 801 kD sized product, the predictedsize of the BTK-C protein if it was translated from the first of the twoadditional start codons (FIG. 4B). The BTK-C protein levels are low,suggesting that transcription from the Btk-C promoter is weak. However,no BTK-A specific sized product was detected in BT474 cell lysate,although it was readily detected in B-cell lysate (FIG. 4B), indicatingthat expression of the Btk-C transcript is distinct from Btk-Aexpression.

PCR performed with primers designed to amplify the sequence locatedbetween the published Ref seq BTK start and stop codons (BTK internal;Table 1) (FIG. 8 and data not shown), produces a product of the expectedsize from BT474 cDNA. Quantitative PCR (qPCR) analysis detects the BTKtranscript in round 34 of 40 total cycles suggesting that BTK levels inBT474 cells are significantly lower than GAPDH reference transcripts.Interestingly, however, RT-PCR analysis of the full length BTKtranscript indicates that the BTK mRNA expressed in BT474 cells ismissing a PCR primer binding site in the 5′UTR when compared to thatexpressed in B-cells. Although internal primer pairs yield products ofthe expected size, no product is amplified from BT474 cDNA when aforward primer designed to hybridize within the 5′ UTR is used (BTK5′UTR; Table 1) (FIG. 8). This 5′ UTR primer does, however, generate aproduct of the expected size from a positive control cDNA samplegenerated from Namalwa B-cells (FIG. 8). This result suggests that theN-terminal sequence of the BTK transcript in BT474 breast cancer cellsdiffers from the published sequence deduced from B-cell mRNA. The 5′ endof the BTK transcript expressed in BT474 cells was obtained usingRACE-PCR and subsequently analyzed by DNA sequencing. When aligned tothe 5′ end of the published BTK message (BTK-A; accession # U13399) thenucleotide sequence of the transcript isolated from BT474 cells isidentical downstream from position 307, however, the sequences upstreamfrom position 307 diverge significantly (FIG. 11A) indicating that theBTK transcript in BT474 cells has an alternative first exon (FIG. 11B).

Since the BTK transcript found in BT474 cells is identical to anautomated computationally predicted (GNOMON) sequence named BTK-cra-C,we refer to this isoform as BTK-C and the alternative exon, exon 1C(FIG. 11B, C). This transcript has been predicted in genome databases(Levy, et al. 2007) (Griffiths-Jones 2004) and isolated as an expressedsequence tag (EST) from human pheochromocytoma tissue (Yang, Y. et al.2000, unpublished; accession # AV733045) and peripheral bloodmononuclear cells (Kimura, et al. 2006). The portion of the sequencethat is specific to the BTK message expressed in BT474 cells is locatedon the right arm of the X-chromosome, 4,416 by distal from the startsite of exon 1 of the published BTK gene (BTK-A). The 5′ end of BTK-C is255 by from the start site of the ribosomal protein L36a gene which istranscribed in the opposite direction (FIG. 11C). A putativetranscription start site exists within a CpG island 200 by upstream fromthe start of exon 1C (Down and Hubbard 2002). In addition, predictedtranscription factor binding sites are also present, including Ets, Ap2,AhR and HoxA7 binding sites (Matys, et al. 2003) (Table 3).Transcription factor binding sites (TFBSs) were predicted for 1000 bpsof genomic sequence located proximal to the first nucleotide of theBTK-C transcript. TFBSs were predicted for the BTK-C promoter using highstringency parameters to minimize false positives (Matys et al., 2003).From the left column, gene symbol for each transcription factor (TF), inthe location of the predicted TFBS in relation to the most 5′-nucleotidetranscribed in the BTK-C mRNA, and the sequence of the predicted TFBSare shown (Table 3). T Capitalization depicts nucleotides that areconserved with consensus TFBS, lower case depicts the adjacent BTK-Cpromoter sequence. Taken together, these data indicate that expressionof the BTK-C transcript in BT474 breast cancer cells is driven by analternate promoter located upstream from the published BTK-A promoter.The first exons from the BTK-A and BTK-C isoforms utilize differentdonor sites to splice into a common acceptor site, located within exon2, to yield the mature BTK-A and BTK-C mRNA isoforms (FIG. 11B).

Due to the additional sequence, the BTK-C message encodes a product thatcontains an amino-terminal 34 amino acid extension to the BTK-A protein(FIG. 12A). This extension is phylogenetically conserved, since DNAsequence encoding it appears in most mammalian species upstream of theBTK start site in each organism (data not shown). A product consistentwith this size is observed on immunoblots as an 80 kD product incellular lysates from several breast cell lines using a polyclonalantibody raised against the pleckstrin homology domain, residues 2-172,of BTK (BD Transduction Laboratory, 611116). The observed size agreeswith the BTK-C predicted size of 79.9 kDa and is larger than the 76.3kDa BTK-A product which is also detected in the breast lines but atsignificantly lower levels than in the control NAMALWA B cell lysate(FIG. 12B). These results indicate that initiation of translation occursfrom the initial methionine codon found in the 5′ end of the BTK-Ctranscript in breast cells. Consistent with this, BTK-A and BTK-Csequences cloned into a retroviral vector containing a CMV promoter anda C-terminal triple flag tag sequence (BTK-A-flag and BTK-C-flagvectors, respectively) produce different sized products. 293FT cellstransiently transfected with the BTK-A flag vector yield a 79.5 kDamolecular weight product, which is in agreement with the predicted sizeof the BTK-A protein containing a triple flag tag (FIG. 17A). When 293FTcells were transiently transfected with the BTK-C flag vector, however,two products are detected of approximately 79 kDa and 83 kDa,respectively (FIG. 12C, FIG. 17A, B). These translation product sizescould result from the BTK-C transcript if translation was initiated fromthe N-terminal methionine (83 kDa) of BTK-C, as well as the methioninestart codon of the BTK-A message which is retained as the 35th codon ofBTK-C (79.5 kDa) (FIG. 11B). shRNAs targeting internal exons of the BTKgene effectively silence the BTK gene decreasing protein expression, aswould be predicted.

The Btk-C Transcript Encodes an Alternative Protein. To engineer more ofthe BTK-C protein, the Btk-C sequence beginning with the regioncorresponding to the new start codon and continuing to the stop codonwas cloned into a Hygro-MarxIV over-expression vector (Hannon et al.,1999) containing a triple flag tag sequence (hereafter referred to asthe Btk-C-flag vector). For comparison, the Btk-A sequence, beginningwith the published start codon and continuing to the stop codon, wasalso cloned into the triple flag tag Hygro-MarxIV vector (hereafterreferred to as the Btk-A-flag vector). 293FT cells were co-transfectedwith the Btk-A-flag vector or Btk-Cflag vector as well as either the BtkshRNA construct or a control shRNA construct. The 293FT cells containingthe over-expressed Btk-A protein alone or with the control shRNA yieldeda 79.5 KD molecular weight product; the predicted size of the Btk-Aprotein containing a triple flag tag (FIG. 4C). The 293FT cellscontaining the over-expressed Btk-C protein alone or with the controlshRNA, however, yielded two products. The smaller product isapproximately the predicted molecular weight of the Btk-A proteincontaining a triple flag tag (79.5 KD) and the larger product is thepredicted molecular weight of the Btk-C protein containing a triple flagtag if it were translated from the first of the two novel methioninecodons (83 KD) (FIG. 4C). Without intending to limit the invention inany embodiment by any theory as to how the embodiment works, Applicantsbelieve that the most likely explanation for the two Btk-C products isthat the first of the two additional methionine codons is being used asa translational start site as well as the original methionine startcodon, which contains a good Kozak consensus sequence. Interestingly,data from western blotting, qPCR and 5′RACE (FIG. 4B and data not shown)indicate that only the Btk-C transcript is present in BT474 cells,suggesting that the Btk-A promoter is not active in BT474 cells.

293FT cells stably over-expressing either the BTK-A or BTK-C proteinsthat were transfected with the shRNA targeting Btk containedsignificantly less cross reactive protein compared to cells transfectedwith the control shRNA (FIG. 4C; FIG. 17A). Additionally, the transienttransfection of BT474 cells stably over-expressing Btk-C with siRNAstargeting Btk resulted in an approximate 70% decrease in Btk proteincompared to control (FIG. 4D; FIG. 17B). That the BTK isoform isimportant to breast cells was confirmed by designing siRNAs that wouldspecifically target this isoform. As shown in FIG. 12B, siRNAscorresponding to exon 1C reduce BTK-C-flag protein expression intransfected HEK 293T cells. Importantly, these siRNAs also decrease theviability of BT474 cells, indicating that this isoform is important forcell viability. Taken together these results confirm that the Btk shRNAand siRNA are strong and specific effectors of Btk gene silencing andthat the expression of this isoform is important for breast cancer cellsurvival.

To assess BTK activation in BT474 cells the phosphorylation status oftyrosine residue number 223, which becomes auto-phosphorylated afteractivation, was assessed. BT474 cells stably over-expressing either theBtk-A-flag or Btk-C-flag proteins were subjected to immunoprecipitationusing a flag specific antibody and the immunoprecipitates were separatedwith SDS-PAGE electrophoresis. Blots were probed with an anti-phosphoTyr²²³-Btk antibody or a total Btk antibody (Santa Cruz, E-9) to controlfor loading. The Btk-A protein was phosphorylated as well as both formsof the Btk-C proteins, indicating the Btk-C protein is activated inBT474 cells (FIG. 5A). Addition of the Btk specific inhibitor LFM-A13severely impeded phosphorylation of both the Btk-A and Btk-C proteins,indicating that auto-phosphorylation of the elongated Btk-C protein isinhibited to a similar level as Btk-A using the Btk-A specific inhibitorLFM-A13 (FIG. 5A). Consistent with this, inhibiting Btk phosphorylationin BT474 cells using 25 uM LFM-A13 increased apoptosis levels by 8%compared to control, further establishing the protective role that Btkplays in BT474 cell survival (FIG. 5B, C). There was no significantchange in growth rate or morphology in BT474 cells stablyover-expressing the BTK-A or the BTK-C proteins compared to controlcells under either standard conditions or serum-free conditions. Thus,although decreased expression results in apoptosis, Btk over-expressiondoes not confer a growth advantage under these conditions (data notshown).

The specific phosphorylation of the BTK-A protein in addition to bothforms of BTK produced from the BTK-C vector, indicate that the BTK-Cprotein is activated in BT474 cells (FIG. 13A) under standard growthconditions. Auto-phosphorylation of BTK was tested by the addition ofthe BTK specific inhibitor LFM-A13 which has an IC50 for BTK of 17 μM(Vassilev and Uckun 2004). Treatment with 35 μM LFM-A13 for 48 hsignificantly impedes phosphorylation of both the BTK-A and BTK-Cproteins, indicating that auto-phosphorylation of the elongated BTK-Cprotein is inhibited to a similar level as BTK-A under these conditions(FIG. 13A). Consistent with this finding, inhibition of BTKauto-phosphorylation in BT474 cells using 35 μM LFM-A13 increasesapoptosis levels six fold compared to control, further establishing theprotective role that BTK plays in BT474 cell survival (FIG. 13B).

Btk is detected in BT474 cell cytoplasm using immunofluorescence.Immunofluorescent (IF) confocal images were taken of wild type BT474cells, BT474 cells containing either a stably integrated controlHygro-MarxIV triple flag tag vector (hereafter referred to as controlvector), the Btk-A-flag vector or the Btk-C flag vector. As wasexpected, no BTK specific signal was generated using a flag tag specificantibody in wt BT474 cells or cells stably over-expressing the controlvector, but a signal was seen in the cytoplasm of both cell lines stablyover-expressing either the BTK-Aflag or the BTK-C-flag proteins (FIG.6B). However, IF images taken of cells probed with a BTK specificantibody were positive for BTK in the cytoplasm of wtBT474 cells (FIG.6A) as well as in the cytoplasm of cells stably over-expressing thecontrol vector (FIG. 6B). As would be expected, cells stablyover-expressing either the BTK-A or BTK-C proteins contained, noticeablymore signal than control cells. The endogenous BTK protein was mostlikely more visible using immunofluorescent confocal imagery becausecertain antibodies are more amenable to immunofluorescent confocalimaging protocols compared to SDS-PAGE immunoblotting.

The data from RACE-PCR and RT-PCR (FIG. 11A, FIG. 9) suggest that BTK-Cmay be preferentially expressed in breast cancer cells compared tonon-tumorigenic cells. Immunohistochemical staining of breast tissuemicroarray samples (Biomax.us BRC-961) shows increased expression of BTKin clinical breast cancer tissues compared to matched, non-tumorigenic,breast tissues using a BTK specific antibody. Significant levels of antiBTK staining are observed in most of the tumor samples (80.2%).Representative images are shown in FIG. 14A. This confirms that at leastsome form of BTK is unexpectedly expressed in cells of this tissue type,although isoform-specific protein level determination is not currentlypossible. So that we could discriminate between BTK-A and BTK-C levelsin normal and cancer cells, isoform specific qPCR primer sets weredesigned to the heterogeneous regions of the two sequences locatedwithin the 5′UTRs (BTK-A_(—)5′UTR and BTK-C_(—)5′UTR, respectively).cDNA from the BTK-A positive B-cell line Namalwa, the breast cancer celllines BT474, MCF7, and MDA-MB-361, the non-tumorigenic breast cell lineMCF10a and human mammary epithelial cells (HMEC), was independentlyamplified with each primer set using SYBR Green. A product is detectedonly for the BTK-A positive malignant B cell line Namalwa using theBTK-A specific primer set (data not shown). However, products aredetected in all tested breast samples using the BTK-C specific primerset. Both the BT474 and MCF7 breast cancer cell lines have 4-fold moreBTK-C transcript compared to either the non-tumorigenic MCF10a and HMECbreast cells or to the malignant B-cell line Namalwa, while MDA-MB-361cells had approximately 2 fold more expression compared to MCF10a orHMEC cells (FIG. 14B).

Although BTK-C expression was higher in each of the cancer cell linestested, isoform-specific expression determinations in tissue sample RNAwas more complex. Ten of 23 breast cancer samples had significantlyhigher expression of BTK-C compared to BTK-A (FIG. 14C). Most of theother breast cancer samples had relatively low levels of both isoforms,with only one tumor sample showing higher levels of BTK-A compared toBTK-C. These results agree with mRNA expression profiling studiesperformed on clinical breast cancer tissues and made available in theOncomine database (Oncomine, 2004) In these studies, BTK-A and BTK-Cisoforms were not considered independently. In the TCGA data set, themedian BTK expression was upregulated 2-fold in invasive breastcarcinoma, 1.85 fold in invasive breast ductal carcinomas and 9-fold ininvasive lobular breast carcinoma compared to normal breast tissue (TheCancer Genome Atlas, 2011). In a second study, total BTK levels areelevated 6.86 fold (p-value=6.50E-6) in the stroma of invasive ductalcancers (7 samples) compared to all matched, cancer-free, breast tissuesamples analyzed (15 samples) (Karnoub, et al. 2007). In another studythat profiled the mRNA expression levels of 54 breast cancer samples to9 non-pathogenic tissue samples, BTK expression was 5.4 fold higher inbreast carcinomas (5 samples), 3.2 fold higher in invasive ductal breastcarcinoma samples (32 samples), and 4.2 fold higher in invasive lobularbreast carcinoma samples (7 samples) compared to non-pathogenic tissuesamples (9 samples) (Radvanyi, et al. 2005). Taken together theseresults indicate that BTK-C expression is enhanced in breast cancercells compared to non-tumorigenic breast cells, further supporting thenotion that expression of the BTK-C transcript, through use of analternative promoter, contributes to the survival of these cells.

Despite decades of study in hematopoiesis, relatively little isunderstood about the effectors of BTK. To explore potential mechanismsof BTK-C in breast cell survival, over-expression constructs were usedto determine if the BTK-A and BTK-C isoforms are functionally distinct.Assessments of growth rate, morphology, or resistance to apoptoticagents in BT474 cells stably over-expressing either the BTK-A or theBTK-C form proteins, under either standard growth conditions orserum-free conditions reveal no significant differences (data notshown). Furthermore, a bioinformatic analysis (ExPASy Proteomics Tools)of exon 1C to identify conserved motifs, including cleavage sites,phosphorylation, N-glycosylation, or N-myristoylation sites does notidentify any putative functional roles.

Since BTK activity has been shown to affect the nuclear localization andactivation of a number of transcription factors in hematopoietic cells,we performed microarray analysis of MCF-10A cells over-expressing theBTK-C isoform. In these experiments, BTK-C expression from a retrovirusCMV promoter is increased approximately eightfold compared to vector(data not shown). Microarray analysis of MCF-10A expressing either theBTK-A or BTK-C isoforms indicates that BTK may also affecttranscriptional targets in breast cells. In these experiments,expression from a retrovirus CMV promoter of either BTK isoform wasincreased approximately eightfold more than vector. One gene upregulatedin cells over-expressing BTK-C is STEAP4 which participates in a widerange of biologic processes (Gomes, et al. 2012; Grunewald, et al.2012), such as control of cell proliferation and apoptosis, and glucoseuptake (Qin, et al. 2011). Since BTK-C also has similar effects onproliferation and apoptosis resistance, we tested whether BTK-C activitywas correlated with glucose uptake by assaying 2-NBDG fluorescence.LFM-A13 inhibits glucose uptake in those breast cancer cell lines thatexpress BTK-C (FIG. 15A, B). Over-expression of BTK-C in MCF-10A cellsalso results in increased glucose uptake that is inhibited by LFM-A13(FIG. 15C, D). These results are consistent with the notion that BTK, byaltering the expression of STEAP4, has the potential to influence bothtumor chemoresistance and energy metabolism, both of which are keyfeatures of the cancer cell phenotype.

Btk-C is elevated in breast cancer cells. The data from westernblotting, 5′RACE and RT PCR suggested that the Btk-C message might bepreferentially expressed in breast cancer cells compared tonon-tumorigenic cells. To specifically amplify the Btk-A and Btk-Cmessages two distinct qPCR primer sets were designed to the uniqueregion of the sequences located within the 5′UTRs, (Btk-A 5′UTR andBtk-CS′UTR, respectively). cDNA from the Btk-A positive B cell line(Namalwa), the breast cancer cell lines BT474, MCF7, and MDA-MB361, aswell as the non-tumorigenic breast cell lines MCF10a and HMEC, wasamplified with each primer set using SYBR Green. A product was detectedonly for the Btk-A positive malignant B cell line Namalwa using theBtk-A specific primer set (data not shown). Products were detected inall breast cancer samples using the Btk-C specific primer set. Thenon-tumorigenic breast samples produced a signal inconsistently and atthe last round of cycling, suggesting the transcript levels were at thelimit of detection. Both the BT474 and MCF7 breast cancer cell lines had4-fold more transcript compared to the either of the non-tumorigenicbreast cell lines MCF10a and HMEC and the malignant B-cell line Namalwa(FIG. 7A) These results indicate Btk-C expression is enhanced in breastcancer cells compared to non-tumorigenic breast cells. While not wishingto be bound by any theory of how embodiments of the invention work, thisresult raises the possibility that mis-expression of the Btk-Ctranscript, through use of an alternative promoter, may support theunregulated growth characteristic to malignant cells.

A search for Btk expression in clinical breast cancer tissues (Oncomine)revealed that Btk levels are elevated in forty three percent (seventotal samples) of invasive ductal cancers compared to all fifteenmatched, cancer-free breast tissue samples analyzed (Karnoub et al.,2007). In a second study that profiled the expression levels of 198breast cancer samples, Btk expression was upregulated in 13% of tissuesamples from patients with invasive ductal breast cancer (Desmedt etal., 2007). Although, the Affymetrix probes used to target the Btk genein these studies does not discriminate between the Btk-A and the Btk-Cforms, based upon our data we would predict that the Btk C form is beingexpressed in these clinical cancer samples. Consistent with the oncomineexpression data, using a BTK specific antibody, BTK was detected in aclinical breast cancer tissue sample but not in a matchednon-tumorigenic breast tissue sample (FIG. 7B).

Using an unbiased RNAi approach to screen 91% of the human genomes PTKs,we have found that 29% of the total TKs examined strongly contributed tothe proliferative potential of the breast cancer cell. Among these TKs,54% were receptor TK's and 46% were non-receptor cytoplasmic tyrosinekinases As expected, known survival kinases such as EGFR, ERBB2, FGFR2,LYN, PTK2B, NTRK2/TRK-B were identified in the screen. EGFR and ERBB-2are known critical survival kinases and ErbB-2 is amplified andconstitutively activated in the BT474 breast cancer cell line. ERBB2 hasno known ligand but rather becomes activated through dimerization withother EGFR family members resulting in constitutive signaling cascadesthrough PLCgamma, PI3K and RAS (FIG. pPLCg2 blot; (Serra et al., 2008);(Eckert et al., 2004).

Additionally, we have revealed previously unrecognized roles for membersof the Eph family of receptor tyrosine kinases and Tec family ofcytoplasmic tyrosine kinases in promoting breast cancer cell survival inthis Erb-B2 positive breast cancer. Four of the 25 TKs that caused thegreatest inhibition of BT474 cellular proliferation when knocked downwere Eph receptor TKs (FIG. 1; Table 2). Eph receptors and Eph ligandshave been well studied for their role in neuronal development (Klein,2004). Additional functions have been described including involvement invascular development during embryogenesis, in cell to cell communicationand in the regulation of cellular morphogenesis, adhesion and migration(Arvanitis and Davy, 2008); (Merlos-Suarez and Batlle, 2008) (Noren andPasquale, 2004). Interestingly, Eph Receptors are also expressed onplatelets and have been implicated in platelet aggregation at sites ofvascular injury (Prevost et al., 2003).

The formation of breast carcinomas is accompanied by the recruitment ofa “variety of stromal cells (such as MSCs) with both pro- andanti-tumorigenic activities” (Karnoub et al., 2007); (Bissell andRadisky, 2001). The response is similar to wound healing and scarformation, and involves the continuous deposition of growth factors,cytokines and matrix-remodeling proteins, such that a tumor site is likea ‘wound that never heals’ (Park et al., 2000). Similarly, both sites ofvascular injury and sites of tumor initiation lead to the formation ofthrombus; the process by which collagen or thrombin activate freelycirculating platelets, leading to their adherence at the injured walland then to each other, resulting in the formation of a fibril clot(Prevost et al., 2005).

The Eph kinase receptors EphA4 and EphB1 are expressed on platelets(Prevost et al., 2005) and Eph receptor interaction with ligand promotesadhesion and aggregation, at sites of vascular injury, in a Ras familymember, Rapl, at least partially, dependent event (Prevost et al.,2005); (Prevost et al., 2004). Furthermore, Eph receptors are known toassociate with Src family tyrosine kinases and to signal throughcytoplasmic tyrosine kinases (Kullander and Klein, 2002). Followingplatelet activation EphA4 becomes associated with the Src familycytoplasmic TKs Lyn and Fyn and may promote the phosphorylation ofintegrin B3 (Prevost et al., 2002). Lyn is another TK integral to B cellreceptor signaling and, when knocked down, led to a significant decreasein BT474 cellular proliferation (FIG. 1; Table 2). Without wishing tosuggest that embodiments of the invention work according to anyparticular mechanism or theory, it is interesting that EGF promoteswound healing (Hardwicke et al., 2008), suggesting a potentiallycooperative or shared signaling pathway exists for thesereceptor/ligands (Lo et al., 2006). Further studies will need to beconducted to determine if EGF and/or EGF receptor family memberscooperate with the Eph receptors to promote breast cancer cell survival.

In addition, we found that knockdown of four of the five Tec familymember kinases resulted in reduced BT474 proliferation (FIG. 1; Table2). The Tec kinases are known primarily for their roles in immunedevelopment and function. Yet, further evaluation of one family member,BTK, led to the discovery of novel protein containing an amino-terminalextension and two additional start codons. A search of the EST databaseusing sequence specific to the Btk-C transcript retrieved two identicalEST sequences verifying that this gene is actively transcribed from analternative promoter five thousand nucleotides downstream from the Btk-Apromoter. Applicants will not be bound by any theory of how embodimentsof the invention work. However, expression of Btk-C but not Btk-A in anumber of breast cancer cells but not in non-tumorigenic cells suggeststhat deregulation of the promoter is responsible for its expression inthe cancer cells. In support of this notion, Btk levels are elevated inseveral ductal carcinoma tissue samples compared to all normal breasttissue samples analyzed in a study represented in the cancer geneexpression database, Oncomine (Karnoub, 2007, Nature). Furtherexperiments will need to be done to determine if the Btk-C variant is infact the form that is elevated in these breast carcinomas.

A number of recent papers provide data that is consistent with ourresults implicating hematopoietic associated cytoplasmic TKs in criticalfunctions in solid tumors. ABL2 is a cytoplasmic TK, highly similar tothe Src and Tec family of cytoplasmic TKS, whose constitutiveactivation, generated through chromosomal translocation into breakpointcluster regions (BCR-Abl) and Tel genes (Tel-Abl), (Advani andPendergast, 2002) causes various forms of leukemia andmyeloproliferative diseases (Tefferi and Gilliland, 2007). Recently,however, Abl has been implicated, for the first time, in breast cancercell pathogenic processes (Srinivasan and Plattner, 2006). Abl was foundto be constitutively active downstream of deregulated ErbB receptors andSrc family tyrosine kinases in highly invasive breast cancer cell lines(Srinivasan and Plattner, 2006) (Srinivasan et al., 2008).

In PTEN negative prostate cancer cell lines, LNCaP and PC3, knockdown ofthe Tec family kinase BMX using siRNAs caused suppression of cellgrowth. BMX was found to be activated by the ErbB2/ErbB3 receptors andthe EGF receptor in a PI3-K dependent and independent manner,respectively. An interaction was identified between BMX and ErbB-3 usingimmunoprecipitation and immunoblotting. Furthermore, the cytoplasmictyrosine kinase Src was shown to be responsible for the phosphorylationof BMX prior to membrane recruitment as a Src inhibitor blocked itsactivation. The authors propose that BMX has a role in integrating thePI3-K and ErbB2/ErbB3 signaling pathways (Jiang et al., 2007). We alsonoted cell death of BT474 cells after Src knockdown (data not shown),suggesting (without wishing to be bound by theory or hypothesis) thatBtk and the other Tec family tyrosine kinases may serve a similarfunction in ErbB-2 positive breast cancers.

To determine how the RANK and Immune Receptor (ITAMs) signaling pathwaysconverge to promote osteoclast differentiation a genome-wide screen ofthe non-receptor tyrosine kinases revealed that osteoclasts, but notosteoblasts, express high levels of Btk and Tec. Osteoclasts are derivedfrom bone marrow cells and are under the control of the immune system.The authors conclude (without intending that embodiments of theinvention must work according to the hypothesis) that RANKL stimulatesthe Btk and Tee kinases to form a signaling complex with othermolecules, such as the adaptor protein BLNK and the tyrosine kinase Syk,which leads to PLCgamma phosphorylation and the induction of calciumsignaling essential for osteoclastogenesis (Shinohara et al., 2008).

Do recruited cell types, such as mesenchymal stem cells, associate withprimary tumor cells in such a way to stimulate Btk and other Tec familytyrosine kinases, leading to a convergence of signaling pathways thatfavor cell survival? Applicants pose this question without admitting inany way that embodiments of the invention work according to thehypothesis implied by the question. In any event, the EGF and BCRsignaling complexes and regulated downstream signaling pathways areremarkably similar (Donjerkovic and Scott, 2000); (Lo et al., 2006).Both involve signaling through PLCgamma, PI3K and RAS with the resultantcalcium flux and subsequent activation of MAPK/JNK. PLCgamma activationleads to the hydrolysis of PIP2 and the production of DG and IP3. DGinduces/phosphorylates PKC leading to the activation of ELK1 and IP3leads to calcium mobilization. The resulting cellular message ispro-survival. An RNAi screen conducted to identify tyrosine kinases andphosphotases that would sensitize chemoresistant cancer cells toapoptosis found a number of calcium-regulated kinases (CaMK1 g,CaMKIINa, CaMKIIB and CaMKIId) to be potent survival kinases (MacKeiganet al., 2005), suggesting that kinases that regulate calcium flux may beimportant therapeutic targets.

We have previously described a novel isoform of the cytosolic,non-receptor tyrosine kinase, Bruton's tyrosine kinase that is essentialfor the survival of breast cancer cells. Here we show that we haveidentified short interfering RNAs that specifically target this isoformand cause the death of breast cancer cells. Since this isoform ispreferentially expressed in cancer cells these siRNAs may representpotential therapeutics.

siRNAs that Specifically Target BTK-C.

Previous work is shown that down regulation of BTK with RNAi orinhibition with pharmacological inhibitors causes apoptosis in breastcancer cells. Overexpression gives rise to increased resistance toapoptosis. Our results also show that BTK has increased expression inseveral breast cancer cell lines and in human breast tumors. Thepredominant BTK protein found in tumors is an alternative form of thekinase which contains an amino-terminal extension. That a novel isoformof this kinase is expressed and is critical for cell survival indicatesthat it may represent a potential therapeutic target for the treatmentof breast cancer.

That the BTK isoform is important to breast cells was confirmed bydesigning siRNAs that would specifically target this isoform. BTK-Cspecific siRNAs were custom synthesized (Dharmacon, Lafayette, CO, USA):siRNA1 sense: GGUUAUUGGAUGCCCAUUAUU (SEQ ID NO: 66), antisense:UAAUGGGCAUCCAAUAACCUU (SEQ ID NO: 67); siRNA2 sense:CAACAAAUGGUUAUUGGAUUU (SEQ ID NO: 68); antisense: AUCCAAUAACCAUUUGUUGUU(SEQ ID NO: 69). As shown in the figure, siRNAs corresponding to exon 1C reduce BTK-C-flag protein expression in transfected HEK 293 T cells.Importantly, these siRNAs also decrease the viability of BT474 cells,indicating that this isoform is important for cell viability (FIG. 12D).

BTK-C Inhibits Apoptosis Induced by Doxorubicin in Breast Cancer Cells.

BTK over-expression has been implicated in imatinib resistance tochronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL)(Villuendas, et al. 2006); (Hofmann, et al. 2002). Its constitutiveactivation due to deregulated B cell receptor (BCR) engagement is anintegral component to certain B cell lymphomas (Irish, et al. 2006);(Kuppers 2005) and it has been shown to serve a protective role throughinhibition of Fas/APO-1 mediated apoptosis (Qiu and Kung 2000; Vassilev,et al. 1999). For this reason we determined whether BTK-C inhibitsapoptosis induced by Doxorubicin in breast cancer cells. The BTK-Cisoform is expressed at relatively low levels in MCF-10A cells (FIG.13C, 14B). Over-expression of BTK-C in MCF-10A cells using Flag-taggedMCF-10-vector (10A-Vec) or MCF-10A-Btk-C (10A-Btk-C) constructs revealsthat BTK-C counteracts the effects of doxorubicin. The number ofapoptotic cells after doxorubicin treatment decreases nearly threefoldin cells over-expressing BTK-C as assayed by cleaved caspase-3 signal(FIG. 13D, E). Treatment of MCF-10A cells over-expressing BTK-C with 35μM LFM-A13 for 24 abolishes the acquired resistance to Doxorubicin (1μM). These results suggest that BTK expression in tumor cells may leadto chemotherapeutic resistance.

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EXPERIMENTAL

Cell culture. Breast cancer cell lines NAMALWA, BT474, MCF-7, SK-BR-3,MDA-MB-361 and MCF10a were obtained from the ATCC. The Burkitt'sLymphoma cell line NAMALWA was obtained from ATCC. Human mammaryepithelial cells (HMEC) were obtained from Cambrex. HEK 293FT cells wereobtained from Invitrogen. BT474, MCF-7 and HEK 293FT cells were culturedin DMEM (Hyclone) supplemented with 10% FBS (Hyclone) and 100 U/μl ofpenicillin-streptomycin (Cellgro). NAMALWA were cultured in RPMI-1640medium (ATCC) supplemented with 10% FBS (Hyclone) and 100 U/μl ofpenicillin-streptomycin. MDA-MB-361 were cultured in RPMI-1640 medium(ATCC) supplemented with 20% FBS and 100 U/0 of penicillin-streptomycin.HMECs were cultured in MEGM medium (Cambrex). MCF10a were cultured inDME/F12 1:1 medium supplemented with 5% Horse serum, 20 ng/ml EGF, 0.5μg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 μg/ml insulin, and 100U/μl of penicillin-streptomycin.

Reagents. The polyclonal anti-BTK antibody (C-20), the monoclonalanti-BTK antibody (E-9) and the polyclonal anti-GAPDH antibody (V-18)were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif., USA).The polyclonal anti-BTK antibody used for immunofluorescence wasobtained from ProSci Incorporated (Poway, Calif., USA). The monoclonalanti-FLAG M2 antibody was from Stratagene (Cedar Creek, Tex., USA). Thepolyclonal anti-caspase-3 antibody (Asp175) was obtained from CellSignaling Technology (Danvers, Mass., USA). The protease inhibitorcocktail was obtained from Roche (Indianapolis, Ind., USA) and thephosphatase inhibitor cocktail was from Pierce (Rockford, Ill., USA).

Construction of the PTK shRNA Library. A collection of short hairpinRNAs (shRNAs) targeting each of the PTKs were assembled from thepShagMagic2 (pSM2) shRNA library (Paddison et al., 2004; Silva et al.2005). pSM2 shRNAs are modeled after pre-miRNAs and are transcribed by apol III type promoter (U6) in a retroviral backbone. These vectors canbe used to transfect cells to induce transient gene knockdown or theycan be used to generate virus capable of infecting cells for long-term,continuous hairpin expression. In most cases, multiple shRNA constructstarget the same gene, such that, over 300 shRNA clones were selectedfrom the library to transfect into BT474 cells. Plasmid DNA wasisolated, in 96-well format, from bacterial stocks containing each ofthe shRNAs using the Perfectprep Plasmid 96 Vac Direct Bind kit(Eppendorf, Hamburg, Germany).

RNAi screen-transfections. shRNA constructs were expressed from thepSHAGMAGIC 2 (pSM2) vector and derived from a genome-wide shRNA library(31). ShRNAs targeting the firefly (Photinus pyralis) luciferase genewere used as controls. Transfection efficiency was monitored byco-transfection with a modified MSCV-Puro vector expressing greenfluorescent protein (GFP). The alamarBlue (Biosource) assay wasperformed 96 h post-transfection, since BT474 cells have a populationdoubling time of ˜100 hours. Mature sequences of the shRNAs thatproduced the best results on decreasing BT474 viability are given inTable 1; a complete list is available in the RNAi Codex web page(codex.cshl.edu). The shRNAs targeting the luciferase gene wereconstructed as described in the RNAi Codex web page(codex.cshl.edu/scripts/newmain.pl) using a modified pSM2 vectorcontaining the PheS gene (pSM2-PheS) in the cloning site, as a negativeselection marker. Quantification of alamarBlue we used a BioTek HTSynergy plate reader. Transfections were performed using FuGENE 6(Roche) according to the manufacturer's protocol. High-throughputtransfections were performed using an EpMotion 5070 fluidics station(Eppendorf). Z-scores were calculated using the following formula:(normalized sample value−normalized data set mean)/data set standarddeviation.

BTK was also knocked down using the siGEMOME SMART pool duplex(Dharmacon, Lafayette, CO, USA) transfected with Oligofectamine Reagent(Invitrogen, Gaithersburg, MD, USA) according to the manufacturer'sinstructions. BTK-C specific siRNAs were custom synthesized (Dharmacon,Lafayette, CO, USA): siRNA1 sense: GGUUAUUGGAUGCCCAUUAUU (SEQ ID NO:66), antisense: UAAUGGGCAUCCAAUAACCUU (SEQ ID NO: 67); siRNA2 sense:CAACAAAUGGUUAUUGGAUUU (SEQ ID NO: 68); antisense: AUCCAAUAACCAUUUGUUGUU(SEQ ID NO: 69).

Cell viability-apoptosis assays. For high-throughput experiments, cellsgrown on 96-well plates were washed once with 1×PBS, fixed with 2.5%formaldehyde and stained with Hoechst 33342 (MolecularProbes-Invitrogen). Cell images were acquired using an In Cell Analyzer1000 (GE Healthcare) high content imaging system, with a 20× objective.At least 50 fields were imaged per single experiment. Cell counts andstatistics were then performed using the In Cell Investigator 3.4high-content image analysis software (GE Healthcare). Apoptosis wasdetected by cleaved Caspase-3 after 48 h to 96 h of shRNA treatments.Apoptosis was detected by cleaved Caspase-3 after 48 h of siRNAtreatments or treatment with the BTK specific inhibitor LFM-A13. BT474cells were treated with 35 μM LFM-A13. Control cells were treated withDMSO. For the cleaved caspase-3 assy, cells were fixed after treatmentwith 2.5% formaldehyde, washed with 1×PBS, permeabilized with 0.1%Triton-X 100 (Fisher Chemicals), blocked with 3% normal goat serum(Sigma-Aldrich), incubated with a 1:50-1:200 dilution of the primaryantibody, washed with 1×PBS, incubated with a 1:800 dilution of thesecondary antibody, washed again with 1×PBS and finally stained withHoechst 33342 (Molecular Probes-Invitrogen). Cells were imaged by the InCell Analyzer 1000 (GE Healthcare) or by a Leica TCS SP5 confocalmicroscope system (Leica Microsystems). At least 500 cells were countedfor cleaved Caspase-3. Apoptotic cells were calculated as a percentageof the total cellular population. Antibodies used: cleaved Caspase-3(Asp175, #9661; Cell Signaling Technology), Alexa Fluor 568 goatanti-rabbit IgG (#A-11011; Invitrogen) and Alexa Fluor 568 goatanti-mouse IgG (#A-11004; Invitrogen), and, Alexa Fluor 568 goatanti-rabbit IgG (#A-11011; Invitrogen).

Immunoblotting. Cell extracts for western blots (immunoblots) wereobtained using RIPA buffer (1% Triton X-100, 40 mM NaCl, 0.1% SDS, 10 mMTris pH 8.0) or non-denaturing lysis buffer: (20 mM Tris (pH 8.0), 137mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA), supplemented withcomplete cocktail of proteinase inhibitors (Roche). BT474 cellscontaining the stably integrated BTK-A-flag tag MarxIV, the BTK-C-flagtag MarxIV, and the 3-galactosidase MarxIV vectors were incubated with100 μM LFM-A13 for 45 min. For detection of phoshporylated epitopes, thePhosSTOP cocktail of phospatase inhibitors (Roche) was added in thelysis buffer. Tyrosine-phosphorylated BTK was assessed byimmunoprecipitation (IP) using anti-Flag and Western blot (WB) analysisusing anti-BTK Phospho (pY223) and anti-BTK. Protein extracts wereseparated by SDS-PAGE, transferred to Immobilon-P (Millipore) membranesand immunoblotted according to standard protocols. Blots were imagedusing a Fluor Chem HD (Alpha Innotech) imaging system. A polyclonalanti-BTK antibody (C-20), the monoclonal anti-BTK antibody (E-9) and apolyclonal anti-GAPDH antibody (V-18) were obtained from Santa CruzBiotechnology (Santa Cruz, Calif., USA). A polyclonal anti-BTK antibodyraised against residues 2-172, of BTK was obtained from Becton Dickinson(BD Transduction Laboratory, 611116). A polyclonal anti-BTK antibodyused for immunofluorescence was obtained from ProSci Incorporated(Poway, Calif., USA). The monoclonal anti-FLAG M2 antibody was fromStratagene (Cedar Creek, Tex., USA). The polyclonal anti-caspase-3antibody (Asp175) was obtained from Cell Signaling Technology (Danvers,Mass., USA). The protease inhibitor cocktail was obtained from Roche(Indianapolis, Ind., USA) and the phosphatase inhibitor cocktail wasfrom Pierce (Rockford, Ill., USA). Antibodies used included: GAPDH(V-18; Santa Cruz Biotechnology), anti-rabbit IgG-HRP (sc2204, SantaCruz Biotechnology), antigoat IgG-HRP (sc-2768, Santa CruzBiotechnology), anti-mouse IgG-HRP (#31430; Pierce Biotechnology).

RNA isolation; quantitative polymerase chain reaction (qpcr);TaqMan-qPCR; RTPCR.

Total RNA was extracted from cells using TRizol (Invitrogen) accordingto the manufacturer's instructions, followed by the addition of DNaseI(Roche) for 20 min at 37° C. and purified using the RNeasy column(Qiagen, Valencia, Calif., USA) cleanup protocol. The cDNA was amplifiedusing a modified version of the NIH/NCl Reid Lab cDNA synthesisprotocol. A mixture containing 2-3.5 μg tRNA, 3 ul oligodT (Promega,Madison, Wis., USA) and 0.5 mM dNTPs was incubated at 65° C. for 5minutes. Following incubation, 1×M-MLV reverse transcriptase buffer(Promega), and 256 U RNase Inhibitor (Fisher Scientific, Pittsburgh,Pa., USA), were added to the tRNA mixture and was incubated at 42° C.for 1 minute. 200u of M-MLV reverse transcriptase (Promega) was thenadded to the tRNA mixture and incubated at 42° C. for 1 hour, followedby a 15 minute incubation at 70° C. to inactivate the M-MLV enzyme. qPCRreactions using SYBR Green Master Mix (Applied Biosystems) or Taq SYBRGreen Super Mix (BioRad), or TaqMan qPCR using TaqMan Gene ExpressionMaster Mix (Applied Biosystems) were performed on a ABI PRISM 7900HTSequence Detectin System (Applied Biosystems). TaqMan qPCR wereperformed using TaqMan Gene Expression Master Mix (Applied Biosystems)on a ABI PRISM 7900HT Sequence Detectin System (Applied Biosystems). Theprimer pairs used were designed using ABI's Primer Express software andare shown in Table 1. After the initial denaturation step (95° C. for2.5 min), PCR reactions consisted of 40 cycles of a 95° C.-15 sec step,and a 60° C.-1 min step. Analysis was conducted using ABsystemsReal-Time Analysis software Version 2.2. The RT-PCR amplification mixconsisted of 1×Taq polymerase buffer (Fisher), 0.2 mM dNTPs, 0.2 uM FwdPrimer, 0.2 uM Rvs Primer (Table 1), 1/10th total volume cDNA (10 μl),and 5u Taq polymerase (Fisher) in a 100 μl total volume. After theinitial denaturation step (95° C. for 2 min), PCR reactions consisted of40 cycles of a 95° C.-30 sec step, 55° C.-30 sec step and a 72° C.-3 minstep. Aliquots of each PCR reaction were electrophoresed on 1% agarosegels. The GeneRacer Kit (Invitrogen) was used, according to themanufacturer's specifications, for amplification of the N-terminalportion of the BTK message.

Measurement of BTK mRNA expression in normal and tumor tissues wasperformed using an array of first-strand complementary DNA (cDNA) fromhuman breast tissues contained in the TissueScan Cancer Survey Panel in384-well plates from OriGene (Rockville, Md.) (CSRT302). The cDNAs wereprepared from normal breast tissues or breast adenocarcinoma biopsysamples. The cDNAs from one plate were used for measurement of BTK mRNAlevels by real-time RT-PCR analysis. The same cDNAs in another platewere used for measurement of actin. The data presented are relative BTKisoform mRNA levels normalized to actin. This experiment was conductedtwice with a representative dataset shown.

Small interfering RNA methodology. Btk was knocked down using thesiGEMOME SMART pool duplex (Dharmacon, Lafayette, Colo., USA)transfected with Oligofectamine Reagent (Invitrogen, Gaithersburg, Md.,USA) according to the manufacturer's instructions.

MarxIV triple flag tag vector construction. The triple flag tag sequencewas amplified using the following reaction conditions; 100 ng of thepCMV-3Tag-3a Vector (Stratagene) as template, 1×Taq polymerase buffer(Fisher), 0.2 mM dNTPs, 0.2 uM each of the pCMV-3× Flag Fwd and Rvsprimers (Table 1), and 5u Taq polymerase (Fisher). The PCR products werepurified using spin columns (LPS inc.). The PCR DNA as well as theMarxIV vector DNA were double digested with 10u ApaI and 10u XhoIrestriction endonucleases (NEB) in 1×NEB buffer #4 containing 1×BSA. Thedigested DNA was run on a 2% agarose gel and the desired DNA fragmentswere cut out and purified using the GeneClean Turbo kit (Qbiogene),according to the manufacturer's specifications. The double digested PCRflag tag product was ligated into the double digested MarxIV vector inusing ApaI and XhoI restriction sites and 1× Ligase buffer (NEB), with400 U Ligase. The ligase mix was transformed in 5-alpha competent E.coli cells (NEB) and plated on LB plates containing 100 μg/mL ampicillin(Amp). Colonies were picked and grown in LB+100 μg/mL Amp overnight forplasmid DNA preparations. Plasmid DNA was double digested with 10u Xhoand 10u ApaI and run on a 2% agarose gel to determine which coloniescontain the MarxIV vector with the integrated triple flag tag sequence.The BTK-A and BTK-C sequences were amplified using a proofreading Taqpolymerase (Phusion DNA polymerase, NEB), according to the manufacturersspecifications.

Construction of the Btk-A and Btk-C MarxIV and MarxIV triple flag tagvectors. The Btk-A sequence was amplified using Namalwa cDNA (10 ul) asa template with the Btk-Flag primer set (Table 1). The Btk-C sequencewas amplified using over-lap extension PCR (OLE). BTK-C(N-terminus) wasamplified using 10 ng pCR2.1-TOPO plasmid DNA (Invitrogen) containingthe N-terminal Btk-C sequence (constructed in the 5′RACE experiment) astemplate with the N-term-BTK-C primer set (Table 1). The amplificationreaction conditions were 0.5 mM of each of the N-term-Btk-C primers(Table 1), 1× Phusion DNA polymerase buffer (NEB), 0.2 mM dNTPs and 2uPhusion DNA polymerase (NEB). The second amplification reactionamplified the C-terminal portion of the Btk-C gene using similarconditions as above except rather than plasmid DNA, Namalwa cDNA wasused as template and the Btk-C-terminus primer set (Table 1) was usedfor amplification in 100μl total volume. BTK-C (full-length) wasamplified using the BTK-C(N-terminus) and BTK-C(C-terminus) PCR DNAs astemplate with the BTK-C-Flag primer set. The BTK-C (full-length) PCRproduct was cloned into the MarxIV-Flag vector using BamHI and XhoI(NEB) restriction sites. The BTK-A PCR product was cloned into theMarxIV-Flag vector using MfeI and XhoI (NEB) restriction sites. Selectedclones were sequence-verified. PCR products resulting from theseamplification reactions were purified using Uprep Spin columns (LPSinc.) and both were subsequently used in a third amplification reactionto generate a PCR product of the complete Btk-C sequence. Theamplification reaction conditions were N-terminus PCR product,C-terminus PCR product, 0.5 mM each of the Btk-C-Flag primer set, 1×Phusion DNA polymerase buffer, 0.2 mM dNTPs and 2u Phusion DNApolymerase. The full-length Btk-C PCR product as well as the MarxIVtriple flag vector DNA were Uprep column purified (LPS inc.) anddouble-digested using 10u BamHI and 10u XhoI restriction endonucleases.The Btk-A PCR product was double-digested using 10u MfeI and 10u XhoIrestriction endonucleases. The digested DNA was run on a 1% agarose geland the desired DNA fragments were cut out and purified using theGeneClean Turbo kit (Qbiogene), according to the manufacturer'sspecifications. Each of the double-digested Btk-A and Btk-C PCR productswere ligated into the double-digested MarxIV triple flag tag vectorusing 1× Ligase buffer (NEB), with 400 U Ligase. The ligase mix wastransformed in competent E. coli cells and plated on LB platescontaining ampicillin (100 μg/mL Amp) Colonies were picked and grown inLB+100 μg/mL Amp overnight for plasmid DNA preparations. Plasmid DNA wasdouble digested with 10u Xho and 10u BamHI (Btk-C) insert or 10u XhoIand 10u MfeI (Btk-A) insert and run on a 1% agarose gel to determinewhich colonies contained the Btk-A or Btk-C DNA fragment within theMarxIV triple flag tag vector. Selected clones were sequence verified.

Stable infections and selection. BT474 cells that over-expressMarxIV-Flag, Btk-A MarxIV-Flag or Btk-C-MarxIV-Flag were selected with75 μg/ml Hygromycin B (Roche Diagnostics) for 10 days after infectionwith retrovirus produced by Phoenix A cells, transfected with either theMarxIV-Flag, Btk-A-Flag or Btk-C-Flag.

5′RACE. Total RNA was extracted from BT474 cells using TRizol(Invitrogen), according to the manufacturer's instructions. The tRNA wasthen incubated with DNase1 (Roche) for 20 min at 37° C., and thenpurified using RNeasy (Qiagen, Valencia, Calif., USA) RNA Cleanupprotocol. The GeneRacer Kit (Invitrogen) was used, according to themanufacturer's specifications, for amplification of the N-terminalportion of the Btk message. Briefly, the 5′ Cap was removed fromfull-length mRNAs. The GeneRacer Oligo was ligated to the message RNAs(mRNAs). The mRNA was reverse transcribed into cDNA. The Btk specifictranscript was amplified in a first round of amplification using theGeneRacer 5′ Primer (complementary to the GeneRacer Oligo sequence) andthe Btk-RACE Primer (Table 1). In a second round of amplification 1 ulof the product from the first amplification reaction was used assubstrate with the Btk-RACE-Nest 3′ primer and the GeneRacer 5′ Nestedprimer (Table 1). The product was gel extracted and ligated into thePCR2.1-TOPO vector. The inserted DNA fragment was sequence verified.

BTK Immunolocalization. Cells were immunostained on cover slips withanti-BTK antibody (ProSci); anti-Flag antibody (Stratagene) and Alexa568conjugated secondary antibody, with Hoechst to stain nuclei, and wereimaged using a Leica TCS SP5 confocal microscope system (LeicaMicrosystems Inc., Bannockburn, Ill., USA). Breast cancer tissue arrayswere obtained from Biomax.us (BRC-961) and contained 96 breast cancercases with a range of disease stages and patient ages. Arrays wereprocessed with standard immunohistochemical procedures. Briefly, slideswere baked at 65° C. for 1 hour, then de-parrafinized in HistoChoiceclearing agent, and rehydrated through a series of decreasingconcentrations of ethanol (100, 95, 70, 50%) and finally into PBS.Slides were washed with PBS/0.3% Triton X-100 for 10 minutes andepitopes retrieved in a pressure cooker for 20 minutes. Sections werethen blocked in 10% donkey serum/3% BSA in PBS and then incubated 0/N at4° C. with BTK antibody (1:200 dillution in 3% BSA/PBS). Slides werewashed in PBS and primary antibody detected with Cy5 donkey anti-mousesecondary antibody (1:250 dilution in 3% BSA/PBS, JacksonImmunologicals). Slides were washed, stained in DAPI to visualize thenuclei, rinsed, and mounted with DABCO antifade in 90% glycerol. TMAcores were imaged on a Zeiss AxioImager Z1 equipped for epifluorescencewith at 300W Zenon exicitation source, 20×0.8 n.a. objective, filtersets optimized for DAP1 and CY5 (Semrock) and a Hammamatsu ORCA digitalCCD camera. Exposure for DAP1 and CY5 was fixed across the tissue arraysample, and autofocusing was used after pre-setting coordinates forimage acquisition using the AxioVision software (Carl Zeiss).

TABLE 1 SEQ SEQ Gene Primer Name ID Fwd Primer ID RVs Primer BTKBtk-Full  4 5′-TGAACTCCAGAAAGAAGAAGGTATG-3′  55′-CCTCCCCTCCCATCTTTATG-3′ BTK Btk-Internal  65′-CGTCTTCTCCCCAACTGAAG-3′  7 5′-TTTGAAAGTGGGACGCTCAT-3′ BTK-ABtk-A-specific  8 5′-TGCTGCGATCGAGTCCCAC-3′  95′-TTTGAAAGTGGGACGCTCAT-3′ BTK-C Btk-C-specific 105′-AAATGGTTATTGGATGCCCATT-3′ 11 5′-TTTGAAAGTGGGACGCTCAT-3′ BTK-CqPCR-Btk-C-specific 12 5′-AAATGGTTATTGGATGCCCATT-3′ 135′-TCTTCTTTCTGGAGTTCACCTGTCT-3′ BTK-A Taq-Btk-A-specific 145′-TCCTTCCTCTCTGGACTGTAAGAATAT-3′ 15 5′-ACTTGGAAGGTGGGACTCGAT-3′Taq-Btk-A-specific 16 5′-56-FAM/TCTCCAGGGCCAGTGTCTGCTGC/ Probe36-TAMSp-3′ BTK-C Taq-Btk-C-specific 17 5′-TCTTTTGGTGGACTCTGCTACGT-3′ 185′-GGCCAGAGGATCTGGGAAA-3′ Taq-Btk-C-specific 195′-56-FAM/TGGCGTTCAGTGAAGGGAGCAGTGT/ Probe 36-TAMSp-3′ Flag tagpCMV-3X Flag 20 5′-GTGAACCGTCAGATCCGCTA-3′ 215′-AGGTACGGGCCCCTATTTATCGTCATC ATCTTTGT-3′ BTK-A BTK-A-Flag 225′-AAAGAACAATTGATGGCCGCAGTGATTCTGGA 23 5′-CTTATCTCGAGGGATTCTTCATCCATGG-3′ ACATC-3′ BTK-C N-term-Btk-C 245′-AGAGTGGATCCGCCACCCTTTATCTCTTTTGGTGG 25 5′-CCAGAATCACTGCGGCCAT-3′ACTC-3′ BTK-C C-term-Btk-C 26 5′-ATGGCCGCAGTGATTCTGG-3′ 275′-CTTATCTCGAGGGATTCTTCATCCATG ACATC-3′ BTK-C BTK-C-Flag 285′-AGAGTGGATCCGCCACCCTTTATCTCTTTTGGTG 29 5′-CTTATCTCGAGGGATTCTTCATCCATGGACTC-3′ ACATC-3′ BTK BTK-RACE 30 5′-GAGCTGGTGAATCCACCGCTTCCTTAGTTCTT 315′-CGACTGGAGCACGAGGACACTGA-3′ C-3′ BTK BTK-RACE-Nest 325′-AGTTGGGGAGAAGACGTAGAGAGGCCCTTCAT-3′ 33 5′-GGACACTGACATGGACTGAAGGAGTA-3′

TABLE 2 Tyrosine kinase genes which when silenced result in greater than50% reduction in normalized proliferation. Symbol Name z-score ABL2V-abl Abelson murine leukemia viral oncogene −5.68 homolog 2 BTK Brutonagammaglobulinemia tyrosine kinase −4.95 FES Feline sarcoma oncogene−4.35 NTRK2 Neurotrophic tyrosine kinase, receptor, type 2 −3.95 PTK2BProtein tyrosine kinase 2 beta −3.95 Ptk9 Protein tyrosine kinase 9−3.38 FGFR2 Fibroblast growth factor receptor 2 −3.20 EPHA1 EPH receptorA1 −3.00 LYN V-yes-1 Yamaguchi sarcoma viral related oncogene −2.93homolog FLT3 Fms-related tyrosine kinase 3 −2.78 IGF1R Insulin-likegrowth factor 1 receptor −2.68 EGFR Epidermal growth factor receptor−2.45 JAK1 Janus kinase 1 (a protein tyrosine kinase) −2.25 DDR1Discoidin domain receptor family, member 1 −2.15 TXK TXK tyrosine kinase−2.08 EPHB2 EPH receptor B2 −2.08 EPHA4 EPH receptor A4 −2.05 RYK RYKreceptor-like tyrosine kinase −1.95 PDGF RBPlatelet-derived growthfactor receptor, beta −1.83 polypeptide ERBB2 V-erb-bZ erythroblasticleukemia viral oncogene −1.80 homolog 2 ZAP70 Zeta-chain (TCR)associated protein kinase 70 kDa −1.70 JAK2 Janus kinase 2 (a proteintyrosine kinase) −1.45 BMX BMX non-receptor tyrosine kinase −1.43 TECTec protein tyrosine kinase −1.30 EPHB6 EPH receptor B6 −1.15

TABLE 3 Symbol Location SEQ ID Sequence POU3F2 −902 44 TTATCttaat Pax-6−801 45 accaaattataCGGAAtccat SZF1-1 −785 46 tccatcttaCCCTGg Spzl −69447 tccGGCGGgttttag PPARG −686 48 gttttagggacgtTAACCtagta HNF3beta −66849 taaagAAACAgttca VDR, CAR, −660 50 aaacaGTTCAgaacgtgcaat PXR 1-Oct−573 51 tcctcATTACttg IPF1 −477 52 cctCATTAactc CACD −466 53 ccaCCCCCZF5 −412 54 taGCCCGgtccct HOXA7 −381 55 aGATTGg BRCA1:USF2 −378 56ttgGGTTG Muscle −362 57 tatcgagtcCACACaggag initiator sequences-19CP2/LBP-Ic/LSF −293 58 GCTGGatgttactcg Pax-8 −257 59 catgcttgCGTGAgc AhR−253 60 cttGCGTGagc v-Myb −211 61 gcaAACGGa c-Ets-I(p54) −210 62caaaCGGAAgtat (ETS1) CDP_CR3 −208 63 aacggaagtaTATAG AP-2 −125 64cgCCCTCgggcg Ets −41 65 aCTTCCtc

The invention claimed is:
 1. A method of treating cancer, comprising: a)providing: i) subject with breast cancer, ii) a chemotherapeutic agent,and iii) an inhibitor of a gene encoding a cytoplasmic tyrosine kinase,wherein said inhibitor comprises an interfering double stranded RNAselected from the group consisting of: sense strand having thenucleotide sequence of SEQ ID NO: 66, an antisense strand having thenucleotide sequence of SEQ ID NO: 67, a sense strand having thenucleotide sequence of SEQ ID NO: 68, and an antisense strand having thenucleotide sequence of SEQ ID NO: 69; and b) treating said subject withsaid chemotherapeutic agent and said inhibitor.
 2. The method of claim1, wherein said cytoplasmic tyrosine kinase is Bruton's Tyrosine Kinase.3. The method of claim 1, wherein said cytoplasmic tyrosine kinase is avariant of Bruton's Tyrosine Kinase comprising an amino-terminalextension.
 4. The method of claim 3, wherein said extension comprises anadditional 34 amino acids.
 5. The method of claim 1, wherein saidchemotherapeutic agent comprises Doxorubicin.
 6. The method of claim 1,wherein treating with said chemotherapeutic agent and said inhibitorresults in reduced proliferation of the breast cancer cells within saidsubject.
 7. A method of treating cancer, comprising: a) providing: i)subject with breast cancer, ii) a chemotherapeutic agent, and iii) aninhibitor of a gene encoding a cytoplasmic tyrosine kinase, wherein saidinhibitor comprises a mixture of interfering double stranded RNAscomprising a sense strand having the nucleotide sequence of SEQ ID NOs:66 and 68 and an antisense strand having the nucleotide sequence of SEQID NOs: 67 and 69; and b) treating said subject with saidchemotherapeutic agent and said inhibitor.
 8. The method of claim 1, Amethod of treating cancer, comprising: a) providing: i) a subject withbreast cancer cells, at least some of said breast cancer cellsexhibiting resistance to a chemotherapeutic agent, and ii) an inhibitorof a gene encoding a cytoplasmic tyrosine kinase, wherein said inhibitorcomprises interfering double stranded RNA selected from the groupconsisting of: sense strand having the nucleotide sequence of SEQ ID NO:66, an antisense strand having the nucleotide sequence of SEQ ID NO: 67,a sense strand having the nucleotide sequence of SEQ ID NO: 68, and anantisense strand having the nucleotide sequence of SEQ ID NO: 69, and b)treating said subject with said inhibitor.
 9. The method of claim 8,wherein said cytoplasmic tyrosine kinase is Bruton's Tyrosine Kinase.10. The method of claim 8, wherein said cytoplasmic tyrosine kinase is avariant of Bruton's Tyrosine Kinase comprising an amino-terminalextension.
 11. The method of claim 10, wherein said extension comprisesan additional 34 amino acids.
 12. The method of claim 8, whereintreating with said inhibitor results in reduced proliferation of atleast some of said breast cancer cells within said subject.
 13. Themethod of claim 8, wherein said inhibitor results in reducedproliferation of at least some breast cancer cells within said subjectidentified as resistant to said chemotherapeutic agent.
 14. A method oftreating cancer, comprising: a) providing: i) a subject with breastcancer cells, at least some of said breast cancer cells exhibitingresistance to a chemotherapeutic agent, and ii) an inhibitor of a geneencoding a cytoplasmic tyrosine kinase, wherein said inhibitor comprisesa mixture of interfering double stranded RNAs comprising a sense strandhaving the nucleotide sequence of SEQ ID NOs: 66 and 68 and an antisensestrand having the nucleotide sequence of SEQ ID NOs: 67 and 69, and b)treating said subject with said inhibitor.
 15. A method of treatingcancer, comprising: a) providing: i) a subject with breast cancer, ii) achemotherapeutic agent, and iii) an inhibitor of a gene encoding acytoplasmic tyrosine kinase wherein said inhibitor comprises interferingdouble stranded RNA selected from the group consisting of: sense strandhaving the nucleotide sequence of SEQ lD NO: 66, an antisense strandhaving the nucleotide sequence of SEQ ID NO: 67, a sense strand havingthe nucleotide sequence of SEQ ID NO: 68, and an antisense strand havingthe nucleotide sequence of SEQ ID NO: 69, b) treating said subject withsaid inhibitor; and c) after step b), treating said subject with saidchemotherapeutic.
 16. The method of claim 15, wherein said cytoplasmictyrosine kinase is Bruton's Tyrosine Kinase.
 17. The method of claim 15,wherein said cytoplasmic tyrosine kinase is a variant of Bruton'sTyrosine Kinase comprising an amino-terminal extension.
 18. The methodof claim 17, wherein said extension comprises an additional 34 aminoacids.
 19. The method of claim 15, wherein said chemotherapeutic agentcomprises Doxorubicin.
 20. The method of claim 15, wherein treating withsaid chemotherapeutic agent results in reduced proliferation of thebreast cancer cells within said subject.
 21. The method of claim 15,wherein said inhibitor results in reduced proliferation of the breastcancer cells within said subject.
 22. A method of treating cancer,comprising: a) providing: i) a subject with breast cancer, ii) achemotherapeutic agent, and iii) an inhibitor of a gene encoding acytoplasmic tyrosine kinase, wherein said inhibitor comprises a mixtureof interfering double stranded RNAs comprising a sense strand having thenucleotide sequence of SEQ ID NOs: 66 and 68 and an antisense strandhaving the nucleotide sequence of SEQ ID NOs: 67 and 69, b) treatingsaid subject with said inhibitor; and c) after step b), treating saidsubject with said chemotherapeutic.